Nanoemulsion respiratory syncytial virus (rsv) subunit vaccine

ABSTRACT

The present application relates to the field of immunology, in particular, a vaccine composition of respiratory syncytial virus (RSV) surface proteins, Fusion (F) and Glycoprotein (G) proteins subunit vaccine preferentially mixed with the immune cell targeting and enhancer, nanoemulsion to induce a protective immune response and avoid vaccine-induce disease enhancement.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application No.61/533,062, filed on Sep. 9, 2011, which is specifically incorporated byreference in its entirety.

FIELD OF THE APPLICATION

The present application relates to the field of immunology, inparticular, a nanoemulsion respiratory syncytial virus (RSV) vaccinecomposition comprising at least one RSV immunogen combined with ananoemulsion adjuvant. The RSV immunogen can be any suitable RSVantigen, such as an RSV surface protein, Fusion (F) and Glycoprotein (G)proteins to form a subunit vaccine. The nanoemulsion RSV vaccine inducesa protective immune response and avoids vaccine-induced diseaseenhancement.

BACKGROUND OF THE INVENTION

Respiratory Syncytial Virus (RSV) is a leading cause of seriousrespiratory disease in young children and the elderly worldwide andthere is no vaccine available against this pathogen. Human respiratorysyncytial virus (HRSV) is the most common etiological agent of acutelower respiratory tract disease in infants and can cause repeatedinfections throughout life. It is classified within the genuspneumovirus of the family paramyxoviridae. Like other members of thefamily, HRSV has two major surface glycoproteins (G and F) that playimportant roles in the initial stages of the infectious cycle. The Gprotein mediates attachment of the virus to cell surface receptors,while the F protein promotes fusion of the viral and cellular membranes,allowing entry of the virus ribonucleoprotein into the cell cytoplasm.

Respiratory syncytial virus (RSV) infection commonly results inbronchiolitis and is the leading cause for infant hospitalization in thedeveloped countries. In addition, RSV is increasingly being described asa major pathogen in the elderly, transplant patients, and chronicobstructive pulmonary disease (COPD) patients (Hacking and Hull, 2002).The development of a safe and immunogenic vaccine to address the infantand elderly population presents a unique opportunity.

Previous methods of viral inactivation for vaccine formulation, such asformaldehyde, resulted in enhanced pulmonary disease and mortality.Extensive research into the development of viral vaccines to address RSVhas met with limited success. Some of the major challenges for RSVvaccine development includes, early age of infection, evasion of innateimmunity, failure of natural infection to induce immunity that preventinfection and the demonstration of vaccine-enhance illness coupled withproblems associated with vaccine stability, purity, reproducibility andpotency (Graham, 2011; Swanson and Settembre, 2011).

Approaches have included inactivation or viruses with formalin and thedemonstration of vaccine-induced enhancement of diseases when infectedwith RSV. The observation that formalin inactivated vaccines have showndiseases-enhancement, included showing the skewed immune response thatis important to prevent enhancement are essential for a protectiveimmune response and having F protein its native state to maintainconformational epitopes is essential for the generation of neutralizingantibodies (Krujigen, 2011; Swanson 2011; McLellan et al., 2011). Thedemonstration that formalin-inactivated RSV vaccine diseases enhancementis not attributable to G protein and that G protein antibodies canreduce viral titers and actually protects against diseases enhancementsuggests that G protein can be incorporated into a vaccine candidate(Radu et al, 2010; Haynes et al, 2009; Johnson et al, 2004). The use oflive attenuated vaccines have met with limited success, as the vaccineshave been shown to be minimally immunogenic (Gomez et al 2009). Theutilization of a recombinant viral expressed F and G proteins vaccineshowed reduced immunogenicity associated with low level of antigenexpression, transient level of expression, cellular specificity and thedemonstration that the purified F protein can be structurally immatureand not the appropriate version for eliciting neutralizing antibodies(Singh and Dennis, 2007; Kim et al, 2010). With the use of subunitvaccine, having an optimal level of F protein is critical for inducingthe appropriate immune response, as the subunit vaccines have beenhindered by the inefficient and inappropriate expression of F and Gproteins (Nallet et al., 2009; Huang and Lawlor 2010). The observationthat subunit vaccine containing F protein, even with adjuvant is notcompletely protective and optimal (Langley et al., 2009), suggests thatF protein presentation within its native state in the virion isessential for usage as a vaccine.

Prior teachings related to nanoemulsions are described in U.S. Pat. No.6,015,832, which is directed to methods of inactivating Gram-positivebacteria, a bacterial spore, or Gram-negative bacteria. The methodscomprise contacting the Gram-positive bacteria, bacterial spore, orGram-negative bacteria with a bacteria-inactivating (or bacterial-sporeinactivating) emulsion. U.S. Pat. No. 6,506,803 is directed to methodsof killing or neutralizing microbial agents (e.g., bacterial, virus,spores, fungus, on or in humans using an emulsion. U.S. Pat. No.6,559,189 is directed to methods for decontaminating a sample (human,animal, food, medical device, etc.) comprising contacting the samplewith a nanoemulsion. The nanoemulsion, when contacted with bacteria,virus, fungi, protozoa or spores, kills or disables the pathogens. Theantimicrobial nanoemulsion comprises a quaternary ammonium compound, oneof ethanol/glycerol/PEG, and a surfactant. U.S. Pat. No. 6,635,676 isdirected to two different compositions and methods of decontaminatingsamples by treating a sample with either of the compositions.Composition 1 comprises an emulsion that is antimicrobial againstbacteria, virus, fungi, protozoa, and spores. The emulsions comprise anoil and a quaternary ammonium compound. U.S. Pat. No. 7,314,624 isdirected to methods of inducing an immune response to an immunogencomprising treating a subject via a mucosal surface with a combinationof an immunogen and a nanoemulsion. The nanoemulsion comprises oil,ethanol, a surfactant, a quaternary ammonium compound, and distilledwater. US-2005-0208083 and US-2006-0251684 are directed to nanoemulsionshaving droplets with preferred sizes. US-2007-0054834 is directed tocompositions comprising quaternary ammonium halides and methods of usingthe same to treat infectious conditions. The quaternary ammoniumcompound may be provided as part of an emulsion. US-2007-0036831 and US2011-0200657 are directed to nanoemulsions comprising ananti-inflammatory agent. Other publications that describe nanoemulsionsinclude U.S. Pat. No. 8,226,965 for “Methods of treating fungal, yeastand mold infections;” US 2009-0269394 for “Methods and compositions fortreating onychomycosis;” US 2010-0075914 for “Methods for treatingherpes virus infections;” US 2010-0092526 for “Nanoemulsion therapeuticcompositions and methods of using the same;” US 2010-0226983 for“Compositions for treatment and prevention of acne, methods of makingthe compositions, and methods of use thereof;” US 2012-0171249 for“Compositions for inactivating pathogenic microorganisms, methods ofmaking the compositions, and methods of use thereof;” and US2012-0064136 for “Anti-aging and wrinkle treatment methods usingnanoemulsion compositions.” However, none of these references teach themethods, compositions and kits of the present invention.

In particular, U.S. Pat. No. 7,314,624 describes nanoemulsion vaccines.However, this reference does not teach the ability to induce aprotective immune response to RSV using the immunogen of the invention.

Prior art directed to vaccines includes, for example, U.S. Pat. No.7,731,967 for “Composition for inducing immune response” (Novartis),which describes an antigen/adjuvant complex comprising at least twoadjuvants. U.S. Pat. No. 7,357,936 for “Adjuvant systems and vaccines”(GSK) describes a combination of adjuvant and antigens. U.S. Pat. No.7,323,182 for “Oil in water emulsion containing saponins” (GSK)describes a vaccine composition with an oil/water formulation. U.S. Pat.No. 6,867,000 for “Method of enhancing immune response to herpes”(Wyeth) describes a combination of viral antigens and cytokines (IL12).U.S. Pat. Nos. 6,623,739, 6,372,227, and 6,146,632, all for “Vaccines”(GSK), are directed to an immunogenic composition comprising an antigenand/or antigen composition and an adjuvant consisting of a metabolizableoil and alpha tocopherol in the form of an oil in water emulsion. U.S.Pat. No. 6,451,325 for “Adjuvant formulation comprising a submicron oildroplet emulsion” (Chiron) is directed to an adjuvant compositioncomprising a metabolizable oil, an emulsifying agent, and an antigenicsubstance, wherein the oil and emulsifying agent are present in the formof an oil-in-water emulsion. The adjuvant composition does not containany polyoxypropylene-polyoxyethylene block copolymer; and the antigenicsubstance is not present in the internal phase of the adjuvantcomposition. Finally, US 20040151734 for “Vaccine and method of use”(GSK) describes a method of treating a female human subject sufferingfrom or susceptible to one or more sexually transmitted diseases (STDs).The method comprises administering to a female subject in need thereofan effective amount of a vaccine formulation comprising one or moreantigens derived from or associated with an STD-causing pathogen and anadjuvant.

There remains a need in the art for an effective RSV vaccine and methodsof making and using the same. The present invention satisfies theseneeds.

SUMMARY OF THE INVENTION

The present invention provides a novel approach for delivering andinducing a protective immune response against RSV infection by combiningat least one pivotal immunogenic viral surface antigen, e.g., F and Gproteins, or antigenic fragments thereof, with a delivery and immuneenhancing oil-in-water nanoemulsion. For example, the nanoemulsion RSVsubunit vaccine of the invention induce a Th1 immune response, a Th2immune response, a Th17 immune response, or any combination thereof.

The nanoemulsion RSV subunit vaccine comprises at least one RSVimmunogen, which is RSV F protein, RSV G protein, an immunogenicfragment of RSV F protein, an immunogenic fragment of RSV G protein, orany combination thereof. Additionally, the nanoemulsion RSV subunitvaccine comprises droplets having an average diameter of less than about1000 nm. The nanoemulsion present in the RSV subunit vaccine comprises:(a) an aqueous phase, (b) at least one oil, (c) at least one surfactant,(d) at least one organic solvent, and (e) optionally at least onechelating agent. Preferably the RSV immunogen is present in thenanoemulsion droplets. In another embodiment, the nanoemulsion RSVvaccine may be administered intranasally. In yet another embodiment ofthe invention, the nanoemulsion RSV vaccine lacks an organic solvent.Furthermore, additional adjuvants may be added to the nanoemulsion RSVvaccine.

In another embodiment of the invention, RSV virion particles are alsopresent in the nanoemulsion RSV subunit vaccine. Preferably the RSVvirion particles are present in the nanoemulsion droplets. The RSVvirion particles can be inactivated by the nanoemulsion. In oneembodiment, the RSV viral genome comprises at least one attenuatingmutation.

The nanoemulsion RSV subunit vaccine may be formulated into anypharmaceutically acceptable dosage form, such as a liquid dispersion,gel, aerosol, pulmonary aerosol, nasal aerosol, ointment, cream, orsolid dose.

The RSV surface antigen and/or RSV virion particles can be from anystrain of RSV. In one embodiment, the RSV surface antigen and/or RSVvirion particles are derived from respiratory syncytial virus (RSV)strain L19 (RSV-L19). In another embodiment, the RSV-L19 virus is ahyperproducer of Fusion (F) and Glycoprotein (G) structural proteinsassociated with viral particles. In yet another embodiment, the RSV-L19virus is attenuated human respiratory syncytial virus (HRSV) strain L19.In one embodiment, the vaccine composition comprises a human respiratorysyncytial virus deposited with the American Type Culture Collection(ATCC) as HRSV-L19.

In one embodiment, the RSV surface antigen further comprises at leastone nucleotide modification denoting attenuating phenotypes. In anotherembodiment, the RSV surface antigen or an antigenic fragment thereof ispresent in a fusion protein. The RSV surface antigen can be a peptidefragment of RSV F protein, a peptide fragment of RSV G protein, or anycombination thereof. Additionally, the RSV surface antigen can bemultivalent.

In another embodiment of the invention, there is provided a method forpreparing an immunogenic preparation, whereby the RSV strain, such asHRSV-L19, is genetically engineered with attenuating mutations anddeletions resulting in an attenuating phenotype. The resultingattenuated RSV virus is cultured in an appropriate cell line andharvested. The harvested virus is then purified free from cellular andserum components. The purified virus is then mixed in an acceptablepharmaceutical carrier for use a vaccine composition. Thus, describedare vaccine compositions comprising an RSV viral genome (such as RSVstrain L19) comprising at least one attenuating mutation, preferably incombination with: F protein, G protein, antigenic fragments of F and/orG protein, or any combination thereof. In yet another embodiment, thevaccine compositions comprise an RSV viral genome (such as RSV strainL19) comprising nucleotide modifications denoting attenuatingphenotypes.

In another embodiment of the invention, the vaccine composition is notsystemically toxic to the subject, and produces minimal or noinflammation upon administration. In another embodiment, the subjectundergoes seroconversion after a single administration of the vaccine.

In one embodiment, described is a method for enhancing immunity to humanrespiratory syncytial virus infections comprising administering to asubject a nanoemulsion formulation comprising RSV F and/or G proteinand/or antigenic fragments thereof. Another embodiment of the inventionis directed to a method for inducing an enhanced immunity againstdisease caused by human respiratory syncytial virus comprising the stepof administering to a subject an effective amount of a vaccinecomposition according to the invention. In some embodiments, the subjectcan produce a protective immune response after at least a singleadministration of the nanoemulsion RSV vaccine. In addition, the immuneresponse can be protective against one or more strains of RSV. Theinduction of enhanced immunity to HRSV is dependent upon the presence ofoptimal levels of antigen. Furthermore, the identification of thecritical level of antigen is important for providing a robust immuneresponse. The demonstration that RSV F protein levels are directlycorrelated with the presence and persistence of neutralizing antibodiesand protection against viral challenge, demonstrates that having a viralstrain that produces optimal levels of the critical immunogenic Fprotein expressed in its natural orientation is seminal for usage as avaccine candidate.

In a further embodiment of the invention, RSV F and/or G protein, and/orantigenic fragments thereof, and/or RSV virion particles, areinactivated and adjuvanted with a nanoemulsion formulation to provide anon-infectious and immunogenic virus preparation. The simple mixing of ananoemulsion with a vaccine candidate has been shown to produce bothmucosal and systemic immune response. The mixing of the RSV virionparticles with a nanoemulsion results in discrete antigen particles inthe oil core of the droplet. The antigen is incorporated within the coreand this allows it to be in a free form which promotes the normalantigen conformation.

The RSV vaccines may be formulated as a liquid dispersion, gel, aerosol,pulmonary aerosol, nasal aerosol, ointment, cream, or solid dose. Inaddition, the RSV vaccines may be administered via any pharmaceuticallyacceptable method, such as parenterally, orally, intranasally, orrectally. The parenteral administration can be by intradermal,subcutaneous, intraperitoneal or intramuscular injection.

In another embodiment of the invention, the nanoemulsion RSV vaccinecomposition comprises (a) at least one cationic surfactant and at leastone non-cationic surfactant; (b) at least one cationic surfactant and atleast one non-cationic surfactant, wherein the non-cationic surfactantis a nonionic surfactant; (c) at least one cationic surfactant and atleast one non-cationic surfactant, wherein the non-cationic surfactantis a polysorbate nonionic surfactant, a poloxamer nonionic surfactant,or a combination thereof; (d) at least one cationic surfactant and atleast one nonionic surfactant which is polysorbate 20, polysorbate 80,poloxamer 188, poloxamer 407, or a combination thereof; (e) at least onecationic surfactant and at least one nonionic surfactant which ispolysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407, or acombination thereof, and wherein the nonionic surfactant is present atabout 0.01% to about 5.0%, or at about 0.1% to about 3%; (e) at leastone cationic surfactant and at least one non-cationic surfactant,wherein the non-cationic surfactant is a nonionic surfactant, and thenon-ionic surfactant is present in a concentration of about 0.05% toabout 10%, about 0.05% to about 7.0%, about 0.1% to about 7%, or about0.5% to about 4%; (f) at least one cationic surfactant and at least onea nonionic surfactant, wherein the cationic surfactant is present in aconcentration of about 0.05% to about 2% or about 0.01% to about 2%; or(g) any combination thereof.

In yet another embodiment of the invention, the RSV vaccines compriselow molecular weight chitosan, medium molecular weight chitosan, highmolecular weight chitosan, a glucan, or any combination thereof. The lowmolecular weight chitosan, median molecular weight chitosan, highmolecular weight chitosan, a glucan, or any combination thereof can bepresent in the nanoemulsion.

The foregoing general description and following brief description of thedrawings and the detailed description are exemplary and explanatory andare intended to provide further explanation of the invention as claimed.Other objects, advantages, and novel features will be readily apparentto those skilled in the art from the following detailed description ofthe invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows TEMcross section images of the 20% W₈₀5EC NE with andwithout 30 μg total HA. The panel on the right illustrates that the HAantigens are located in the oil droplets. The darkly stained antigensare located outside of the NE particles.

FIG. 2: Shows endpoint titer of RSV specific IgG in sera of BALB/c miceimmunized with RSV. Only group immunized with 20% W₈₀5EC mixed withF-protein responded to vaccination. The bar represents group average.

FIG. 3: Shows endpoint titer of RSV specific IgG1 (A), IgG2a (B), IgG2b(C), and IgE (D) in sera of BALB/c mice immunized with NE+F ptn. Serawere obtained two weeks after the second immunization.

FIG. 4: Shows the results of vaccination of mice with nanoemulsion(NE)-F-protein attenuates disease following intranasal challenge withlive RSV. Immunized mice were vaccinated intranasally (i.n.) twice atday 0 and day 28 with NE+F-protein, F-protein alone or treated with PBSonly. Control and vaccinated mice were challenged 2 weeks following theboost (i.n.) with 105 PFU live RSV. The expression of virus transcriptswere determined at day 8 post-infection via QPCR of lung RNA.

FIG. 5: Shows that nanoemulsion (NE)-RSV immunization does not promoteimmunopotentiation when compared to non-vaccinated mice. Mice werevaccinated with NE-RSV as described below. Control and vaccinated micewere challenged at day 56. Airway hyperreactivity was assessed at day 8post-challenge via plethysmography. Columns represent the increase inairway resistance following a single, optimized intravenous dose ofmethacholine.

FIG. 6: Shows that inflammation and mucus production in nanoemulsion(NE)+F-protein vaccinated mice does not differ from controls. (A)depicts representative histology (Periodic Acid Schiff's, PAS;Hematoxylin and Eosin, H&E) from control RSV infected and NE+F-Proteinvaccinated mice at day 8 post-infection. Eosinophils were not present.In (B), the expression of Muc5ac and Gob5 were assessed at day 8post-infection via QPCR of lung RNA.

FIG. 7: Shows that nanoemulsion (NE)+F-protein vaccination promotesmixed Th1 and Th2 responses. Mice were vaccinated with NE+F-proteinF-protein alone as described below, and challenged with live RSV. In(A), the expression of IL-12(p40) and (B) IL-17 cytokines were assessedfrom lung RNA via QPCR. In (C) Lung associated lymph node (LALN) cellsuspensions were restimulation with RSV (MOI,0.5). Supernatantscollected for analysis on the Bioplex to assay for cytokine productionin each of the samples.

FIG. 8: Shows F protein units measured followingadministration/immunization with various formulations: (1) 4.5 μg Fprotein, (2) 2.5 μg F protein, (3) 5.5 μL RSV/β-propiolactone (β-PL);(4) 5.6 μL RSV; (5) 0.5 μg F/5.6 μL RSV; (6) 1 μg F/5.6 μLRSV/β-propiolactone (β-PL); and (7) 2.5 μg F/5.6 μL RSV.

FIG. 9: Shows mRNA expression in the lung for IL-4, IL-5, IL-13, IFNγ,IL-17A, and Gob5 following immunization with (1) F protein only; (2) RSVvirus only; and (3) RSV virus+F protein.

FIG. 10: Shows a histological examination of immunized and challengedmice. FIG. 10A shows histological examination of primary RSV infection;FIG. 10B shows histological examination of RSV-NE immunized animal; FIG.10C shows histological examination of F protein immunized animal; andFIG. 10D shows histological examination of RSV+F protein immunizedanimal.

FIG. 11: Shows an SDS PAGE of HRSV Infected Cell Lysate (SDS treated)with L19 and A2.

FIG. 12: Shows an SDS-PAGE of RSV strain L19 and RSV strain A2 HRSV CellLysate (cells & supernatant).

FIG. 13: Shows an SDS PAGE of HRSV strain L19 and strain A2 PurifiedVirus.

FIG. 14: Shows a Western blot of HRSV strain L19 and strain A2 F and GProtein expression 24 hours after Virus Infection.

FIG. 15: Shows the viral inactivation by Western blot assessment, withlanes containing: (1) W₈₀5EC (Lane 1), (2) W₈₀5EC+0.03% B 1,3 Glucan(lane 2), (3) W₈₀5EC+0.3% Chitosan (medium molecular weight)+acetic acid(lane 3), (4) W₈₀5EC+0.3% P407 (lane 4), (5) W₈₀5EC+0.3% Chitosan (lowmolecular weight)+0.1% acetic acid (lane 5), (6) media alone (lane 6);(7) βPL-inactivated virus (lane 7), and (8) L19 positive control (lane9).

FIG. 16: Shows Western blot analysis performed with anti-RSV antibody(anti-G); L19 virus 4×10⁶ PFU/lane, 2×10⁶ PFU/lane, and 1×10⁶PFU/lane+/−βPL inactivation combined with W₈₀5EC as indicated. Specimenswere analyzed fresh (FIG. 16A) or after 14 days at 4° C. or roomtemperature (RT) (FIG. 16B). MM=Molecular weight.

FIG. 17: Shows the immune response (IgG, μg/ml) at week 3 followingvaccination in mice vaccinated IM with different nanoemulsionformulations with and without chitosan: (1) RSV strain L19+2.5%W₈₀5EC+0.1% Low Mol. Wt. Chitosan; (2) RSV strain L19+5% W₈₀5EC; (3) RSVstrain L19+2.5% W₈₀5EC; (4) RSV strain L19+βPL inactivated virus; and(5) naive mice (no vaccine).

FIG. 18: Shows a vaccination schedule for an evaluation of twonanoemulsion-adjuvanted vaccines in cotton rats (Example 13). The twoformulations evaluated include the W₈₀5EC and the W₈₀P₁₈₈5EC (1:1:5)(see Tables 5 and 6 below). Cotton rats received two doses of 30 μl INof the nanoemulsion-adjuvanted vaccine containing 6.6 μg F-ptn. Theywere challenged with 5×10⁵ pfu RSV strain A2 at week 23. Half of theanimals were sacrificed at day 4 and half were sacrificed on day 8.

FIG. 19: Shows the results of an immunogenicity study of W₈₀P1885ECnanoemulsion inactivated RSV vaccine in cotton rats. In the left panel,the Y axis shows the end point titers of specific antibody to F proteinand the X axis shows the time period in weeks. In the right panel theY-axis shows the serum antibody levels in μg/ml and the X-axis shows thetime period in weeks. D4 and D8 show the antibody level in the seraafter the challenge.

FIG. 20: Shows the results of an immunogenicity study of W₈₀5ECnanoemulsion inactivated RSV vaccine in cotton rats. The Y axis showsthe end point titers of specific antibody to F protein and the X axisshows the time period in weeks.

FIG. 21: Shows the immunogenicity of RSV neutralization in cotton rats.Cotton rats were vaccinated with 30 μl of vaccine intranasally, boostedat 4 weeks, and bled at weeks 0, 4, 6, and 8. Study groups included twogroups that received 20% W₈₀5EC nanoemulsion mixed with either 1.6×10⁵PFU RSV strain L19 containing 3.3 μg F protein (n=8) or 3.2×10⁵ PFU RSVstrain L19 containing 6.6 μg F protein (n=8), as well as two groups thatreceived 20% W₈₀P₁₈₈5EC nanoemulsion mixed with either 1.6×10⁵ PFU RSVstrain L19 containing 3.3 μg F protein (n=8) or 3.2×10⁵ PFU RSV strainL19 containing 6.6 μg F protein (n=8). Neutralization units (NEU)represent a reciprocal of the highest dilution that resulted in 50%plaque reduction. NEU measurements were performed at 4 weeks (pre boost)and at 6 weeks (2 weeks post boost). Specimens obtained at 6 weeksgenerated humoral immune responses adequate to allow for NEU analysis.Data is presented as geometric mean with 95% confidence interval (CI)(FIG. 21A). Correlation between EU and NEU is for all animals at 6 weeksusing Spearman rho.

FIG. 22: Shows neutralizing antibodies on day 4 and day 8. FIG. 22Ashows the results for W₈₀P₁₈₈5EC nanoemulsion combined with RSV strainL19, and FIG. 22B shows the results for W₈₀5EC nanoemulsion combinedwith RSV strain L19. All cotton rats demonstrated high neutralizingantibodies (NU) against the vaccine RSV strain L19. Neutralizingantibodies were rising steadily following the challenge (Y axis). Day 8neutralizing units (NU) were higher than Day 4 NU. Naïve Cotton Rats didnot show any neutralization activity in their sera.

FIG. 23: Shows the Specific activity of serum antibodies showed that thespecific activity (Neutralizing units/ELISA units) of the serumantibodies tends to increase on Day 8 when compared to Day 4post-challenge. FIG. 23A shows the results for W₈₀P₁₈₈5EC nanoemulsioncombined with RSV strain L19 (NU/EU for the Y axis), at Day 4 and Day 8.FIG. 23B shows the results for W₈₀5EC nanoemulsion combined with RSVstrain L19 (NU/EU for the Y axis), at Day 4 and Day 8.

FIG. 24: Shows cross protection at Day 4 for cotton rats that received 3doses of RSV L19 adjuvanted vaccine, then challenged with RSV strain A2.

FIG. 24A shows the results for W₈₀P₁₈₈5EC nanoemulsion combined with RSVstrain L19, and FIG. 24B shows the results for W₈₀5EC nanoemulsioncombined with RSV strain L19. Serum neutralization activity showsequivalent NU against RSV strain L19 or RSV strain A2, demonstratingcross protection between the two RSV strains.

FIG. 25: Shows viral clearance (RSV strain A2) at Day 4 in lungs ofCotton Rats. Vaccinated cotton rats (vaccinated with W₈₀P₁₈₈5ECnanoemulsion combined with RSV strain L19, or W₈₀5EC nanoemulsioncombined with RSV strain L19) showed complete clearance of RSV strain A2challenged virus from the lungs of cotton rats. Naïve animals wereshowing >10³ pfu RSV strain A2/gram of lung.

FIG. 26: Shows IM Cotton rat vaccination and challenge schedule.

FIG. 27: Shows Serum immune response in cotton rats vaccinated IM with20% W₈₀5EC nanoemulsion mixed with 1.6×10⁵ PFU RSV strain L19 containing3.3 μg F protein. The Y axis shows serum IgG, μg/mL, over a 14 weekperiod, at day 4 post-challenge, and at day 8 post-challenge.

FIG. 28: Shows Serum immune response in cotton rats vaccinated IM with20% W₈₀5EC nanoemulsion mixed with 1.6×10⁵ PFU RSV strain L19 containing3.3 μg F protein. FIG. 28A shows the end point titers (Y axis) over a 14week period, at day 4 post-challenge, and at day 8 post-challenge. FIG.28B shows the ELISA units (Y axis) over a 14 week period, at day 4post-challenge, and at day 8 post-challenge.

FIG. 29: Shows IM vaccinated cotton rats showed complete clearance ofthe RSV 4 days following the challenge compared to Naïve animals. Showsviral clearance (RSV strain A2) at Day 4 in lungs of Cotton Rats. IMvaccinated cotton rats (vaccinated with W₈₀5EC nanoemulsion combinedwith RSV strain L19) showed complete clearance of RSV strain A2challenged virus from the lungs of cotton rats. Naïve animals wereshowing 10³ pfu RSV strain A2 or greater/gram of lung.

FIG. 30: Shows the measurement of anti-F antibodies (Y axis) over an 8week period (X axis) for mice vaccinated either IM or IN with RSVvaccine containing 2×10⁵ plaque forming units (PFU) of L19 RSV viruswith 1.7 μg of F protein inactivated with 20% W₈₀5EC nanoemulsionadjuvant. BALB/C mice (n=10/arm) were vaccinated at weeks 0 and 4 IN orIM. Serum was analyzed for anti-F antibodies.

FIG. 31: Shows the measurement of RSV-specific cytokines. Cytokines weremeasured in cells from spleens, cervical and intestinal lymph nodes (LN)following vaccination of BALB/C mice (n=10/arm) at weeks 0 and 4 IN orIM with RSV vaccine containing 2×10⁵ plaque forming units (PFU) of L19RSV virus with 1.7 μg of F protein inactivated with 20% W₈₀5ECnanoemulsion adjuvant. Cytokines measured included IFNg, IL-2, IL-4,IL-5, IL-10, and IL-17.

FIG. 32: Shows measurement of the cytokines IL-4, IL-13, and IL-17 inlung tissue following either IN or IM vaccination of BALB/C mice(n=10/arm) at weeks 0 and 4 IN or IM with RSV vaccine containing 2×10⁵plaque forming units (PFU) of L19 RSV virus with 1.7 μg of F proteininactivated with 20% W₈₀5EC nanoemulsion adjuvant. IL-4 and IL-13showing greater expression following IM administration, with IL-17showing greater expression following IN administration.

FIG. 33: Shows the measurement of airway resistance (cm H₂O/mL/sec) inmice following either IN or IM vaccination of BALB/C mice (n=10/arm) atweeks 0 and 4 IN or IM with RSV vaccine containing 2×10⁵ plaque formingunits (PFU) of L19 RSV virus with 1.7 μg of F protein.

DESCRIPTION OF THE INVENTION I. Overview

The present invention provides for the novel formulation of RSV surfaceantigens, F and G proteins mixed with nanoemulsion to address theinadequate immune response observed in previous data of RSV vaccines. Anoptimal vaccine against RSV would not only prevent against acute viralinfection but also prevent against reinfections.

The nanoemulsion RSV subunit vaccine comprises at least one RSVimmunogen, which is RSV F protein, RSV G protein, an immunogenicfragment of RSV F protein, an immunogenic fragment of RSV G protein, orany combination thereof. Additionally, the nanoemulsion RSV subunitvaccine comprises nanoemulsion droplets having an average diameter ofless than about 1000 nm. Preferably the RSV immunogen is present in thenanoemulsion droplets. In another embodiment of the invention, RSVvirion particles are also present in the nanoemulsion RSV subunitvaccine. Preferably the RSV virion particles are present in thenanoemulsion droplets.

The present invention provides a novel approach for delivering andinducing a protective immune response against RSV infection by combininga pivotal immunogenic RSV viral surface antigen, F and/or G proteins,with a delivery and immune enhancing oil-in-water nanoemulsion.Utilization of isolated RSV viral surface antigens shown to be the majorviral immunogens independent from other viral components, such as viralprotein NS1, which can skew the immune response resulting in enhanceddisease, is an important foundation for a subunit vaccine. Further,mixing one or more of the RSV surface antigens with a nanoemulsion,which preferentially encloses the antigens and acts as a delivery systemto the appropriate immune cells and additionally as a potent immuneenhancing component, underscores the novelty of the present invention.Compared to other subunit vaccines and recombinant vaccines with resultslacking for a fully functional human vaccine, the nanoemulsion RSVsubunit viral surface antigens provide significant novelty compared toprevious candidates in its ability to generate a robust, sustainable andprotective immune response.

The induction of enhanced immunity to RSV is dependent upon the presenceand presentation of an optimal level of antigens. Combining isolated RSVsurface antigens with a nanoemulsion provides a novel approach todeliver the vaccine to appropriate antigen presenting cells of theimmune response.

The nanoemulsion compositions of the invention function as a vaccineadjuvant. Adjuvants serve to: (1) bring the antigen—the substance thatstimulates the specific protective immune response—into contact with theimmune system and influence the type of immunity produced, as well asthe quality of the immune response (magnitude or duration); (2) decreasethe toxicity of certain antigens; (3) reduce the amount of antigenneeded for a protective response; (4) reduce the number of dosesrequired for protection; (5) enhance immunity in poorly respondingsubsets of the population and/or (7) provide solubility to some vaccinescomponents.

In one embodiment, multivalent subunit vaccine can be constructedutilizing surface antigens F and G proteins derived from RSV and mixedwith nanoemulsion.

In another embodiment, derivatives and fusions proteins can be designedfrom the RSV surface antigens F and G proteins and are then mixed withnanoemulsion to generate a subunit vaccine.

In one embodiment, subunit vaccines can be constructed with one or moreof RSV surface antigens, namely F and G proteins mixed with ananoemulsion. It is entirely possible to have both F and G proteinsadded together and mixed with a nanoemulsion in a resulting subunitvaccine composition. In another embodiment, either F or G protein mixedwith a nanoemulsion is a suitable subunit vaccine according to theinvention. Antigenic fragments of F and/or G protein can also beutilized in the nanoemulsion RSV vaccines of the invention.

Nanoemulsions are oil-in-water emulsions composed of nanometer sizeddroplets with surfactant(s) at the oil-water interface. Because of theirsize, the nanoemulsion droplets are pinocytosed by dendritic cellstriggering cell maturation and efficient antigen presentation to theimmune system. When mixed with different antigens, nanoemulsionadjuvants elicit and up-modulate strong humoral and cellular T_(H)1-typeresponses as well as mucosal immunity (Makidon et al., “Pre-ClinicalEvaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine,”PLoS ONE. 3(8): 2954; 1-15 (2008); Hamouda et al., “A Novel NanoemulsionAdjuvant Enhancing The Immune Response from Intranasal Influenza Vaccinein Mice in National Foundation for Infectious Disease,” 11th AnnualConference on Vaccine Research. Baltimore, Md. (2008); Myc et al.,“Development of immune response that protects mice from viralpneumonitis after a single intranasal immunization with influenza Avirus and nanoemulsion,” Vaccine, 21(25-26):3801-14 (2003); Bielinska etal., “Mucosal Immunization with a Novel Nanoemulsion-Based RecombinantAnthrax Protective Antigen Vaccine Protects against Bacillus anthracisSpore Challenge,” Infect Immun., 75(8): 4020-9 (2007); Bielinska et al.,“Nasal Immunization with a Recombinant HIV gp120 and NanoemulsionAdjuvant Produces Th1 Polarized Responses and Neutralizing Antibodies toPrimary HIV Type 1 Isolates,” AIDS Research and Human Retroviruses,24(2): 271-81 (2008); Bielinska et al., “A Novel, Killed-Virus NasalVaccinia Virus Vaccine,” Clin. Vaccine Immunol., 15(2): 348-58 (2008);Warren et al., “Pharmacological and Toxicological Studies onCetylpyridinium Chloride, A New Germicide,” J. Pharmacol. Exp. Ther.,74:401-8) (1942)). Examples of such antigens include protective antigen(PA) of anthrax (Bielinska et al., Infect. Immun., 75(8): 4020-9(2007)), whole vaccinia virus (Bielinska et al., Clin. Vaccine Immunol.,15(2): 348-58 (2008)) or gp120 protein of Human Immune Deficiency Virus(Bielinska et al., AIDS Research and Human Retroviruses. 24(2): 271-81(2008)). These studies demonstrate the broad application of thenanoemulsion adjuvant with a variety of antigens including RSV antigens.

In one embodiment of the invention, the nanoemulsion RSV vaccinecomprises droplets having an average diameter of less than about 1000 nmand: (a) an aqueous phase; (b) about 1% oil to about 80% oil; (c) about0.1% to about 50% organic solvent; (d) about 0.001% to about 10% of asurfactant or detergent; or (e) any combination thereof. In anotherembodiment of the invention, the nanoemulsion vaccine comprises: (a) anaqueous phase; (b) about 1% oil to about 80% oil; (c) about 0.1% toabout 50% organic solvent; (d) about 0.001% to about 10% of a surfactantor detergent; and (e) F and G surface antigens of RSV or immunogenicfragments thereof. In another embodiment of the invention, thenanoemulsion lacks an organic solvent.

The quantities of each component present in the nanoemulsion and/ornanoemulsion vaccine refer to a therapeutic nanoemulsion and/ornanoemulsion RSV vaccine.

The methods comprise administering to a subject a nanoemulsion RSVvaccine, wherein the nanoemulsion vaccine comprises droplets having anaverage diameter of less than about 1000 nm. In an exemplary embodimentof the invention, the nanoemulsion RSV vaccine further comprises (a) anaqueous phase, (b) at least one oil, (c) at least one surfactant, (d) atleast one organic solvent, (e) RSV surface antigens, F and G proteins,and (f) optionally comprising at least one chelating agent, or anycombination thereof. In another embodiment of the invention, thenanoemulsion lacks an organic solvent.

In one embodiment, the subject is selected from adults, elderlysubjects, juvenile subjects, infants, high risk subjects, pregnantwomen, and immunocompromised subjects. In another embodiment, thenanoemulsion RSV vaccine may be administered intranasally.

The nanoemulsion RSV subunit vaccine composition can be delivered viaany pharmaceutically acceptable route, such as by intranasal route ofother mucosal routes. Other exemplary pharmaceutically acceptablemethods include intranasal, buccal, sublingual, oral, rectal, ocular,parenteral (intravenously, intradermally, intramuscularly,subcutaneously, intracisternally, intraperitoneally), pulmonary,intravaginal, locally administered, topically administered, topicallyadministered after scarification, mucosally administered, via anaerosol, or via a buccal or nasal spray formulation. Further, thenanoemulsion RSV vaccine can be formulated into any pharmaceuticallyacceptable dosage form, such as a liquid dispersion, gel, aerosol,pulmonary aerosol, nasal aerosol, ointment, cream, semi-solid dosageform, or a suspension. Further, the nanoemulsion RSV vaccine may be acontrolled release formulation, sustained release formulation, immediaterelease formulation, or any combination thereof. Further, thenanoemulsion RSV vaccine may be a transdermal delivery system such as apatch or administered by a pressurized or pneumatic device (i.e., a“gene gun”).

A. RSV Strain L19

In one embodiment of the invention, the RSV strain utilized in thenanoemulsion RSV vaccine is RSV Strain L19. Additionally, the F and/or Gprotein, or antigenic fragment thereof, utilized in the nanoemulsion RSVstrain can be from RSV Strain L19.

It was surprisingly discovered that cells infected with RSV L19 virusproduce between 3-11 fold higher quantities of RSV viral proteins ascompared to cells infected with RSV A2 virus (see Example 6, infra.). Inone embodiment of the invention, the RSV antigen present in the vaccinesof the invention is RSV L19 virus, and more preferably human RSV L19virus, including the purified, attenuated human respiratory syncytialvirus (HRSV) strain L19 (HRSV-L19). In yet other embodiments of theinvention, the RSV viral genome can comprise at least one attenuatingmutation, including but not limited to nucleotide modifications denotingattenuating phenotypes. Additionally, the nanoemulsion RSV vaccine ofthe invention can comprise F or G protein, or antigenic fragmentsthereof, from RSV L19 virus.

RSV L19 strain was found to cause infection and enhanced respiratorydisease (ERD) in mice. Moreover, data published showed that it conferredprotection without induction of ERD in mice when formulated withnanoemulsion.

The RSV Strain L19 isolate was isolated from an RSV-infected infant withrespiratory illness in Ann Arbor, Mich. on 3 Jan. 1967 in WI-38 cellsand passaged in SPAFAS primary chick kidney cells followed by passage inSPAFAS primary chick lung cells prior to transfer to MRC-5 cells(Herlocher 1999) and subsequently Hep2 cells (Lukacs 2006). Comparisonof RSV L19 genome (15,191-nt; GenBank accession number FJ614813) withthe RSV strain A2 (15,222-nt; GenBank accession number M74568) showsthat 98% of the genomes are identical. Most coding differences betweenL19 and A2 are in the F and G genes. Amino acid alignment of the twostrains showed that F protein has 14 (97% identical) and G protein has20 (93% identical) amino acid differences.

RSV L19 strain has been demonstrated in animal models to mimic humaninfection by stimulating mucus production and significant induction ofIL-13 using an inoculum of 1×10⁵ plaque forming units (PFU)/mouse byintra-tracheal administration (Lukacs 2006).

Importantly and uniquely, the RSV L19 viral strain is unique in that itproduces significantly higher yields of F protein (approximately 10-30fold more per PFU) than the other strains. F protein content may be akey factor in immunogenicity and the L19 strain currently elicits themost robust immune response. The L19 strain has a shorter propagationtime and therefore will be more efficient from a manufacturingperspective.

Most significantly, nanoemulsion-inactivated and adjuvanted RSV L19vaccines are highly immunogenic in the universally accepted cotton ratmodel. Cotton rats elicited a rise in antibody titers after oneimmunization and a significant boost after the second immunization(approximately a 10-fold increase). The antibodies generated are highlyeffective in neutralizing live virus and there is a linear relationshipbetween neutralization and antibody titers. Furthermore, antibodiesgenerated in cotton rats showed cross protection when immunized with theRSV L19 strain and challenged with the RSV A2 strain. Both IM and INimmunization established memory that can be invoked or recalled after anexposure to antigen either as a second boost or exposure to live virus.

In another embodiment of the invention, the RSV vaccines of theinvention are cross-reactive against at least one other RSV strain notpresent in the vaccine (or cross-reactive against one or more RSVstrains). As it is known to one of ordinary skill in the art, crossreactivity can be measured 1) using ELISA method to see if the sera fromvaccinated animals or individuals will produce antibodies againststrains that were not used in the administered vaccine; 2) Immune cellswill produce cytokines when stimulated in vitro using stains that werenot used in the administered vaccine. Cross protection can be measuredin vitro when antibodies in sera of animals vaccinated with one strainwill neutralize infectivity of another virus not used in theadministered vaccine.

II. Definitions

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

The term “antigenic fragment” of an RSV surface antigen preferablyrefers to a peptide having at least about 5 consecutive amino acids of anaturally occurring or mutant RSV F protein or RSV G protein. Theantigenic fragment can be any suitable length, such as between about 5amino acids in length up to and including the full length of the F or Gprotein. The F protein is about 518 amino acids in length, and the Gprotein is about 242 amino acids in length. For example, the antigenicfragment can also be about 10, about 15, about 20, about 30, about 40,about 50, about 60, about 70, about 80, about 90, about 100, about 110,etc up to about 242 amino acids in length for a G protein antigenicfragment, and up to about 518 amino acids in length for an F proteinantigenic fragment.

The term “nanoemulsion,” as used herein, includes small oil-in-waterdispersions or droplets, as well as other lipid structures which canform as a result of hydrophobic forces which drive apolar residues(i.e., long hydrocarbon chains) away from water and drive polar headgroups toward water, when a water immiscible oily phase is mixed with anaqueous phase. These other lipid structures include, but are not limitedto, unilamellar, paucilamellar, and multilamellar lipid vesicles,micelles, and lamellar phases. The present invention contemplates thatone skilled in the art will appreciate this distinction when necessaryfor understanding the specific embodiments herein disclosed.

As used herein, the term “antigen” refers to proteins, polypeptides,glycoproteins or derivatives or fragment that can contain one or moreepitopes (linear, conformation, sequential, T-cell) which can elicit animmune response. Antigens can be separated in isolated viral proteins orpeptide derivatives.

As used herein, the term “F protein” refers to a polypeptide or proteinhaving all or partial amino acid sequence of an RSV Fusion protein. Asused herein, F protein includes either F1, F2 or both components of theRSV Fusion protein/polypeptide.

As used herein, the term “G protein” refers to a polypeptide or proteinhaving all or partial amino acid sequence of RSV G attachmentprotein/polypeptide.

As used herein, the term “isolated” refers to proteins, glycoproteins,peptide derivatives or fragment or polynucleotide that is independentfrom its natural location. Viral components that are independentlyobtained through recombinant genetics means typically leads to productsthat are relatively purified.

As used herein, the term “adjuvant” refers to an agent that increasesthe immune response to an antigen (e.g., RSV surface antigens). A usedherein, the term “immune response” refers to a subject's (e.g., a humanor another animal) response by the immune system to immunogens (i.e.,antigens) the subject's immune system recognizes as foreign. Immuneresponses include both cell-mediated immune responses (responsesmediated by antigen-specific T cells and non-specific cells of theimmune system—Th1, Th2, Th17) and humoral immune responses (responsesmediated by antibodies). The term “immune response” encompasses both theinitial “innate immune responses” to an immunogen (e.g., RSV surfaceantigens) as well as memory responses that are a result of “acquiredimmunity.”

As used herein, the term “RSV surface antigens” refers to proteins,glycoproteins and peptide fragments derived from the envelope of RSVviruses. Preferred RSV surface antigens are F and G proteins. The RSVsurface antigens are generally extracted from viral isolates frominfected cell cultures, or produced by synthetically or usingrecombinant DNA methods. The RSV surface antigens can be modified bychemical, genetic or enzymatic means resulting in fusion proteins,peptides, or fragments.

As used herein, the term “immunogen” refers to an antigen that iscapable of eliciting an immune response in a subject. In preferredembodiments, immunogens elicit immunity against the immunogen (e.g., apathogen or a pathogen product) when administered in combination with ananoemulsion of the present invention.

As used herein, the term “enhanced immunity” refers to an increase inthe level of acquired immunity to a given pathogen followingadministration of a vaccine of the present invention relative to thelevel of acquired immunity when a vaccine of the present invention hasnot been administered.

As used herein, the term “virion” refers to isolated, maturedrespiratory syncytial virus particles obtained from infected mammaliancell culture. As used herein, virion can refer to either RSV-1 orRSV-viral particles.

As used herein, the term “multivalent vaccines” refers to a vaccinecomprising more than one antigenic determinant of a single viral agentor multiples strains. As used herein, multivalent vaccine comprisemultiple RSV viral surface antigens, F, F1, F2 and G proteins.Multivalent vaccines could be constructed with antigens derived fromboth RSV-1 and RSV-2.

As used herein, the term “inactivated” RSV refers to virion particlesthat are incapable of infecting host cells and are noninfectious inpertinent animal models.

As used herein, the term “subunit” refers to isolated and generallypurified RSV glycoproteins that are individually or mixed further withnanoemulsion comprising a vaccine composition. The subunit vaccinecomposition is free from mature virions, cells or lysate of cell orvirions. The method of obtaining a viral surface antigen that isincluded in a subunit vaccine can be conducted using standardrecombinant genetics techniques and synthetic methods and with standardpurification protocols.

III. Characteristics of the Nanoemulsion RSV Vaccines

A. Stability

The nanoemulsion RSV vaccines of the invention can be stable at about40° C. and about 75% relative humidity for a time period of at least upto about 2 days, at least up to about 2 weeks, at least up to about 1month, at least up to about 3 months, at least up to about 6 months, atleast up to about 12 months, at least up to about 18 months, at least upto about 2 years, at least up to about 2.5 years, or at least up toabout 3 years.

In another embodiment of the invention, the nanoemulsion RSV vaccines ofthe invention can be stable at about 25° C. and about 60% relativehumidity for a time period of at least up least up to about 2 days, atleast up to about 2 weeks, to about 1 month, at least up to about 3months, at least up to about 6 months, at least up to about 12 months,at least up to about 18 months, at least up to about 2 years, at leastup to about 2.5 years, or at least up to about 3 years, at least up toabout 3.5 years, at least up to about 4 years, at least up to about 4.5years, or at least up to about 5 years.

Further, the nanoemulsion RSV vaccines of the invention can be stable atabout 4° C. for a time period of at least up to about 1 month, at leastup to about 3 months, at least up to about 6 months, at least up toabout 12 months, at least up to about 18 months, at least up to about 2years, at least up to about 2.5 years, at least up to about 3 years, atleast up to about 3.5 years, at least up to about 4 years, at least upto about 4.5 years, at least up to about 5 years, at least up to about5.5 years, at least up to about 6 years, at least up to about 6.5 years,or at least up to about 7 years.

The nanoemulsion RSV vaccines of the invention can be stable at about−20° C. for a time period of at least up to about 1 month, at least upto about 3 months, at least up to about 6 months, at least up to about12 months, at least up to about 18 months, at least up to about 2 years,at least up to about 2.5 years, at least up to about 3 years, at leastup to about 3.5 years, at least up to about 4 years, at least up toabout 4.5 years, at least up to about 5 years, at least up to about 5.5years, at least up to about 6 years, at least up to about 6.5 years, orat least up to about 7 years.

These stability parameters are also applicable to nanoemulsion adjuvantsand/or nanoemulsion RSV vaccines.

B. Immune Response

The immune response of the subject can be measured by determining thetiter and/or presence of antibodies against the RSV immunogen afteradministration of the nanoemulsion RSV vaccine to evaluate the humoralresponse to the immunogen. Seroconversion refers to the development ofspecific antibodies to an immunogen and may be used to evaluate thepresence of a protective immune response. Such antibody-based detectionis often measured using Western blotting or enzyme-linked immunosorbent(ELISA) assays or hemagglutination inhibition assays (HAI). Persons ofskill in the art would readily select and use appropriate detectionmethods.

Another method for determining the subject's immune response is todetermine the cellular immune response, such as throughimmunogen-specific cell responses, such as cytotoxic T lymphocytes, orimmunogen-specific lymphocyte proliferation assay. Additionally,challenge by the pathogen may be used to determine the immune response,either in the subject, or, more likely, in an animal model. A person ofskill in the art would be well versed in the methods of determining theimmune response of a subject and the invention is not limited to anyparticular method.

Experiments conducted during the course of the development of thecurrent invention, demonstrated that nanoemulsion added to hepatitis Bsurface antigen (HBsAg) and administered intranasally was safe andeffective hepatitis B vaccine. The mucosal vaccine induced a Th1associated cellular immune response, with concomitant neutralizingantibodies production. A single nasal immunization of the HBsAgnanoemulsion mixture produces a rapid induction of serum antibodies thatwas comparable to currently administered intramuscular vaccines.Further, there was demonstration of affinity maturation in the antibodyresponse, which is predictive of the potential efficacy of vaccine(Makidon et al., 2008).

Most significantly, as detailed in the examples below, all RSV vaccinesformulated in a nanoemulsion and administered intranasally (IN) orintramuscularly (IM) elicited a protective immune response thatprevented infection of immunized animals. Moreover,nanoemulsion-inactivated and adjuvanted RSV vaccines are highlyimmunogenic in the universally accepted cotton rat model. Cotton ratselicited a rise in antibody titers after one immunization and asignificant boost after the second immunization (approximately a 10-foldincrease). The antibodies generated are highly effective in neutralizinglive virus and there is a linear relationship between neutralization andantibody titers. Furthermore, antibodies generated in cotton rats showedcross protection when immunized with the RSV L19 strain and challengedwith the RSV A2 strain. Both IM and IN immunization established memorythat can be invoked or recalled after an exposure to antigen either as asecond boost or exposure to live virus.

Another emerging component of vaccine protective efficacy is theinduction of T-helper-17 (Th17) cytokine responses. The demonstrationthat IL-17 contributes to the normal immune response to pathogens, hasbeen further utilized to show relevance in vaccination strategies(DeLyrica et al. 2009; Conti et al., 2009). In the development of thecurrent invention, mucosal immunization with nanoemulsion can productadjuvant effects in activating Th1 and Th17 immunity. Mucosalimmunization with nanoemulsion resulted in activation of innate immunewhich directly helps in the induction of Th1 and Th17 cells. The resultsfurther clarify the immune enhancing features of nanoemulsion importancein the field of vaccination for the induction of cellular immunityagainst inactivated RSV virions (Bielinska et al., 2010; Lindell et al,2011).

C. Virus Inactivation

Vaccines need to comprise inactivated virus, particularly when thevaccine comprises whole virus, e.g., to ensure that the vaccine does notcause the disease it is treating and/or preventing. In other words,inactivation of virus ensures that the vaccine does not compriseinfectious particles. Approaches have included inactivation of viruseswith formalin. However, formalin-inactivated vaccines have showndisease-enhancement, including showing a skewed immune response that isimportant to prevent disease-enhancement, and priming by maturedendritic cells, which are essential for a protective immune response.The use of live attenuated vaccines has met with limited success, as thevaccines have been shown to be minimally immunogenic.

In the methods and compositions of the invention, the nanoemulsionfunctions to inactivate and adjuvant the whole virus and/or viralantigens to provide a non-infectious and immunogenic virus.Alternatively, the virus (whole or antigens) can be inactivated prior tocombining with the nanoemulsion. Examples of chemical methods of viralinactivation include, but are not limited to, formalin orβ-propiolactone (β-PL), physical methods of viral inactivation includeusing heat or irradiation, or by molecular genetics means to produce anon-infectious particles. The simple mixing of a nanoemulsion with avaccine candidate has been shown to produce both mucosal and systemimmune response. The mixing of the RSV virion particles with ananoemulsion results in discrete antigen particles in the oil core ofthe droplet. The antigen is incorporated within the core and this allowsit to be in a free form which promotes the normal antigen conformation.

IV. Nanoemulsion RSV Vaccines

A. RSV Immunogen

The RSV immunogen present in the nanoemulsion RSV vaccines of theinvention is an RSV surface antigen, such as F protein, G protein,and/or antigenic fragments thereof. The F protein, G protein andantigenic fragments thereof can be obtained from any known RSV strain.Additionally, the RSV vaccine can comprise whole RSV virus, includingnative, recombinant, and mutant strains of RSV, which is combined withthe one or more RSV antigens. In one embodiment of the invention, theRSV virus can be resistant to one or more antiviral drugs, such asresistant to acyclovir. Any known RSV strain can be used in the vaccinesof the invention. The nanoemulsion RSV vaccines can comprise RSV wholevirus from more than one strain of RSV, as well as RSV antigens frommore than one strain of RSV.

Examples of useful strains of RSV include, but are not limited to, anyRSV strain, including subgroup A and B genotypes, as well as RSV strainsdeposited with the ATCC, such as: (1) Human RSV strain A2, depositedunder ATCC No. VR-1540; (2) Human RSV strain Long, deposited under ATCCNo. VR-26; (3) Bovine RSV strain A 51908, deposited under ATCC No.VR-794; (4) Human RSV strain 9320, deposited under ATCC No. VR-955; (5)Bovine RSV strain 375, deposited under ATCC No. VR-1339; (6) Human RSVstrain B WV/14617/85, deposited under ATCC No. VR-1400; (7) Bovine RSVstrain Iowa (FS1-1), deposited under ATCC No. VR-1485; (8) Caprine RSVstrain GRSV, deposited under ATCC No. VR-1486; (9) Human RSV strain18537, deposited under ATCC No. VR-1580; (10) Human RSV strain A2,deposited under ATCC No. VR-1540P; (11) Human RSV mutant strain A2cpts-248, deposited under ATCC No. VR-2450; (12) Human RSV mutant strainA2 cpts-530/1009, deposited under ATCC No. VR-2451; (13) Human RSVmutant strain A2 cpts-530, deposited under ATCC No. VR-2452; (14) HumanRSV mutant strain A2 cpts-248/955, deposited under ATCC No. VR-2453;(15) Human RSV mutant strain A2 cpts-248/404, deposited under ATCC No.VR-2454; (16) Human RSV mutant strain A2 cpts-530/1030, deposited underATCC No. VR-2455; (17) RSV mutant strain subgroup B cp23 Clone 1A2,deposited under ATCC No. VR-2579; and (18) Human RSV mutant strainSubgroup B, Strain B1, cp52 Clone 2B5, deposited under ATCC No. VR-2542.

Any suitable amount of RSV immunogen can be used in the nanoemulsion RSVvaccines of the invention. For example, the nanoemulsion RSV vaccine cancomprise less than about 100 μg of RSV immunogen (total RSV immunogenand not per RSV immunogen). In another embodiment of the invention, thenanoemulsion RSV vaccine can comprise less than about 90 μg, less thanabout 80 μg, less than about 70 μg, less than about 60 μg, less thanabout 50 μg, less than about 40 μg, less than about 30 μg, less thanabout 20 μg, less than about 15 μg, less than about 10 μg, less thanabout 9 μg, less than about 8 μg, less than about 7 μg, less than about6 μg, less than about 5 μg, less than about 4 μg, less than about 3 μg,less than about 2 μg, or less than about 1 μg of RSV immunogen (totalRSV immunogen and not per RSV immunogen).

In another embodiment of the invention, the RSV vaccines of theinvention comprise about 1.0×10⁵ pfu (plaque forming units (pfu) up toabout 1.0×10⁸ pfu, and any amount in-between, of an RSV virus orantigen. The RSV virus or antigen is inactivated by the presence of thenanoemulsion adjuvant. For example, the RSV vaccines can comprise about1.0×10⁵, 1.1×10⁵, 1.2×10⁵, 1.3×10⁵, 1.4×10⁵, 1.5×10⁵, 1.6×10⁵, 1.7×10⁵,1.8×10⁵, 1.9×10⁵, 2.0×10⁵, 2.1×10⁵, 2.2×10⁵, 2.3×10⁵, 2.4×10⁵, 2.5×10⁵,2.6×10⁵, 2.7×10⁵, 2.8×10⁵, 2.9×10⁵, 3.0×10⁵, 3.1×10⁵, 3.2×10⁵, 3.3×10⁵,3.4×10⁵, 3.5×10⁵, 3.6×10⁵, 3.7×10⁵, 3.8×10⁵, 3.9×10⁵, 4.0×10⁵, 4.1×10⁵,4.2×10⁵, 4.3×10⁵, 4.4×10⁵, 4.5×10⁵, 4.6×10⁵, 4.7×10⁵, 4.8×10⁵, 4.9×10⁵,5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵, 7.5×10⁵, 8.0×10⁵, 8.5×10⁵,9.0×10⁵, 9.5×10⁵, 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶,4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, 7.0×10⁶, 7.5×10⁶,8.0×10⁶, 8.5×10⁶, 9.0×10⁶, 9.5×10⁶, 1.0×10⁷, 1.5×10⁷, 2.0×10⁷, 2.5×10⁷,3.0×10⁷, 3.5×10⁷, 4.0×10⁷, 4.5×10⁷, 5.0×10⁷, 5.5×10⁷, 6.0×10⁷, 6.5×10⁷,7.0×10⁷, 7.5×10⁷, 8.0×10⁷, 8.5×10⁷, 9.0×10⁷, 9.5×10⁷, 1.0×10⁸ pfu of anRSV virus.

In one embodiment of the invention, the RSV vaccines comprise F and/or Gprotein of an RSV strain, such as but not limited to F and/or G proteinof RSV strain L19. In another embodiment, the RSV vaccines compriseabout 0.1 μg up to about 100 μg, and any amount in-between, of RSV Fand/or G protein, such as F and/or G protein of RSV strain L19. Forexample, the RSV vaccines can comprise about 0.1 μg, about 0.2 μg, about0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about0.8 μg, about 0.9 μg, about 1.0 μg, about 1.1 μg, about 1.2 μg, about1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about1.8 μg, about 1.9 μg, about 2.0 μg, about 2.1 μg, about 2.2 μg, about2.3 μg, about 2.4 μg, about 2.5 μg, about 2.6 μg, about 2.7 μg, about2.8 μg, about 2.9 μg, about 3.0 μg, about 3.1 μg, about 3.2 μg, about3.3 μg, about 3.4 μg, about 3.5 μg, about 3.6 μg, about 3.7 μg, about3.8 μg, about 3.9 μg, about 4.0 μg, about 4.1 μg, about 4.2 μg, about4.3 μg, about 4.4 μg, about 4.5 μg, about 4.6 μg, about 4.7 μg, about4.8 μg, about 4.9 μg, about 5.0 μg, about 5.1 μg, about 5.2 μg, about5.3 μg, about 5.4 μg, about 5.5 μg, about 5.6 μg, about 5.7 μg, about5.8 μg, about 5.9 μg, about 6.0 μg, about 6.1 μg, about 6.2 μg, about6.3 μg, about 6.4 μg, about 6.5 μg, about 6.6 μg, about 6.7 μg, about6.8 μg, about 6.9 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg, about8.5 μg, about 9.0 μg, about 9.5 μg, about 10.0 μg, about 10.5 μg, about11.0 μg, about 11.5 μg, about 12.0 μg, about 12.5 μg, about 13.0 μg,about 13.5 μg, about 14.0 μg, about 14.5 μg, about 15.0 μg, about 15.5μg, about 16.0 μg, about 16.5 μg, about 17.0 μg, about 17.5 μg, about18.0 μg, about 18.5 μg, about 19.0 μg, about 19.5 μg, about 20.0 μg,about 21.0 μg, about 22.0 μg, about 23.0 μg, about 24.0 μg, about 25.0μg, about 26.0 μg, about 27.0 μg, about 28.0 μg, about 29.0 μg, about30.0 μg, about 35.0 μg, about 40.0 μg, about 45.0 μg, about 50.0 μg,about 55.0 μg, about 60.0 μg, about 65.0 μg, about 70.0 μg, about 75.0μg, about 80.0 μg, about 85.0 μg, about 90.0 μg, about 95.0 μg, or about100.0 μg of RSV F protein, such as F protein of RSV strain L19.

The RSV immunogen present in the vaccine of the invention can be (1) RSVF protein, (2) RSV G protein; (3) an immunogenic fragment of RSV Fprotein, (4) an immunogenic fragment of RSV G protein; (5) a derivativeof RSV F protein; (6) a derivative of RSV G protein; (7) a fusionprotein comprising RSV F protein or an immunogenic fragment of RSV Fprotein; (8) a fusion protein comprising RSV G protein or an immunogenicfragment of RSV G protein (9) or any combination thereof. Preferably,the RSV vaccine of the invention comprises at least one F proteinimmunogen and at least one G protein immunogen.

In an embodiment of the invention, an immunogenic fragment G protein ofcomprises at least 4 contiguous amino acids of the RSV G protein. Inother embodiments, the RSV G protein fragment comprises about 4, about5, about 10, about 15, about 20, about 25, about 50, about 75, about100, about 125, about 150, about 175, about 200, about 225, about 250,about 275, about 280, about 285, about 289, about 290, about 295, orabout 299 contiguous amino acids of RSV G protein. RSV G glycoproteinhas about 289 to about 299 amino acids (depending on the virus strain).Conservative amino acid substitutions can be made in the G immunogenicprotein fragments to generate G protein derivatives.

In another embodiment of the invention, an immunogenic fragment Fprotein of comprises at least 4 contiguous amino acids of the RSV Fprotein. In other embodiments, the RSV F protein fragment comprisesabout 4, about 5, about 10, about 15, about 20, about 25, about 50,about 75, about 100, about 125, about 150, about 175, about 200, about225, about 250, about 275, about 300, about 325, about 350, about 375,about 400, about 425, about 450, about 475, or about 500 contiguousamino acids of RSV F protein. Conservative amino acid substitutions canbe made in the F immunogenic protein fragments to generate F proteinderivatives.

In some embodiments, the F protein derivatives are immunogenic and havea % identify to the F protein selected from the group consisting of 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%,70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%,56%, 55%, 54%, 53%, 52%, 51%, or 50%. In some embodiments, the G proteinderivatives are immunogenic and have a % identify to the G proteinselected from the group consisting of 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%,78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%,64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or50%.

In one embodiment, a vaccine composition will be constructed withisolated viral surface antigens, F and G proteins combined with isolatedwhole RSV virion particles, which are mixed together with a preferredoil-in-water nanoemulsion.

B. Nanoemulsion

1. Droplet Size

The nanoemulsion RSV vaccine of the present invention comprises dropletshaving an average diameter size of less than about 1,000 nm, less thanabout 950 nm, less than about 900 nm, less than about 850 nm, less thanabout 800 nm, less than about 750 nm, less than about 700 nm, less thanabout 650 nm, less than about 600 nm, less than about 550 nm, less thanabout 500 nm, less than about 450 nm, less than about 400 nm, less thanabout 350 nm, less than about 300 nm, less than about 250 nm, less thanabout 220 nm, less than about 210 nm, less than about 205 nm, less thanabout 200 nm, less than about 195 nm, less than about 190 nm, less thanabout 175 nm, less than about 150 nm, less than about 100 nm, greaterthan about 50 nm, greater than about 70 nm, greater than about 125 nm,or any combination thereof. In one embodiment, the droplets have anaverage diameter size greater than about 125 nm and less than or equalto about 600 nm. In a different embodiment, the droplets have an averagediameter size greater than about 50 nm or greater than about 70 nm, andless than or equal to about 125 nm.

2. Aqueous Phase

The aqueous phase can comprise any type of aqueous phase including, butnot limited to, water (e.g., H₂O, distilled water, purified water, waterfor injection, de-ionized water, tap water) and solutions (e.g.,phosphate buffered saline (PBS) solution). In certain embodiments, theaqueous phase comprises water at a pH of about 4 to 10, preferably about6 to 8. The water can be deionized (hereinafter “DiH₂O”). In someembodiments the aqueous phase comprises phosphate buffered saline (PBS).The aqueous phase may further be sterile and pyrogen free.

3. Organic Solvents

Organic solvents in the nanoemulsion RSV vaccines of the inventioninclude, but are not limited to, C₁-C₁₂ alcohol, diol, triol, dialkylphosphate, tri-alkyl phosphate, such as tri-n-butyl phosphate,semi-synthetic derivatives thereof, and combinations thereof. In oneaspect of the invention, the organic solvent is an alcohol chosen from anonpolar solvent, a polar solvent, a protic solvent, or an aproticsolvent.

Suitable organic solvents for the nanoemulsion RSV vaccine include, butare not limited to, ethanol, methanol, isopropyl alcohol, propanol,octanol, glycerol, medium chain triglycerides, diethyl ether, ethylacetate, acetone, dimethyl sulfoxide (DMSO), acetic acid, n-butanol,butylene glycol, perfumers alcohols, isopropanol, n-propanol, formicacid, propylene glycols, sorbitol, industrial methylated spirit,triacetin, hexane, benzene, toluene, diethyl ether, chloroform,1,4-dixoane, tetrahydrofuran, dichloromethane, acetone, acetonitrile,dimethylformamide, dimethyl sulfoxide, formic acid, polyethylene glycol,an organic phosphate based solvent, semi-synthetic derivatives thereof,and any combination thereof.

4. Oil Phase

The oil in the nanoemulsion RSV vaccine of the invention can be anycosmetically or pharmaceutically acceptable oil. The oil can be volatileor non-volatile, and may be chosen from animal oil, vegetable oil,natural oil, synthetic oil, hydrocarbon oils, silicone oils,semi-synthetic derivatives thereof, and combinations thereof.

Suitable oils include, but are not limited to, mineral oil, squaleneoil, flavor oils, silicon oil, essential oils, water insoluble vitamins,Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate,Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthylanthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate,neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyladipate, C₁₂₋₁₅ alkyl lactates, Cetyl lactate, Lauryl lactate,Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate,Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluidparaffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil,Coconut oil, corn oil, Cottonseed oil, Flaxseed oil, Grape seed oil,Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seedoil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Teaoil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil(simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheatgerm oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil,Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nutoil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniperoil, seed oil, almond seed oil, anise seed oil, celery seed oil, cuminseed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil,cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemongrass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leafoil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmintleaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil,flower oil, chamomile oil, clary sage oil, clove oil, geranium floweroil, hyssop flower oil, jasmine flower oil, lavender flower oil, manukaflower oil, Marhoram flower oil, orange flower oil, rose flower oil,ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil,sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewoodoil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincenseoil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemonpeel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil,valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearylalcohol, semi-synthetic derivatives thereof, and any combinationsthereof.

The oil may further comprise a silicone component, such as a volatilesilicone component, which can be the sole oil in the silicone componentor can be combined with other silicone and non-silicone, volatile andnon-volatile oils. Suitable silicone components include, but are notlimited to, methylphenylpolysiloxane, simethicone, dimethicone,phenyltrimethicone (or an organomodified version thereof), alkylatedderivatives of polymeric silicones, cetyl dimethicone, lauryltrimethicone, hydroxylated derivatives of polymeric silicones, such asdimethiconol, volatile silicone oils, cyclic and linear silicones,cyclomethicone, derivatives of cyclomethicone,hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane,decamethylcyclopentasiloxane, volatile linear dimethylpolysiloxanes,isohexadecane, isoeicosane, isotetracosane, polyisobutene, isooctane,isododecane, semi-synthetic derivatives thereof, and combinationsthereof.

The volatile oil can be the organic solvent, or the volatile oil can bepresent in addition to an organic solvent. Suitable volatile oilsinclude, but are not limited to, a terpene, monoterpene, sesquiterpene,carminative, azulene, menthol, camphor, thujone, thymol, nerol,linalool, limonene, geraniol, perillyl alcohol, nerolidol, farnesol,ylangene, bisabolol, farnesene, ascaridole, chenopodium oil,citronellal, citral, citronellol, chamazulene, yarrow, guaiazulene,chamomile, semi-synthetic derivatives, or combinations thereof.

In one aspect of the invention, the volatile oil in the siliconecomponent is different than the oil in the oil phase.

5. Surfactants

The surfactant in the nanoemulsion RSV vaccine of the invention can be apharmaceutically acceptable ionic surfactant, a pharmaceuticallyacceptable nonionic surfactant, a pharmaceutically acceptable cationicsurfactant, a pharmaceutically acceptable anionic surfactant, or apharmaceutically acceptable zwitterionic surfactant.

Exemplary useful surfactants are described in Applied Surfactants:Principles and Applications. Tharwat F. Tadros, Copyright 8 2005WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30629-3), whichis specifically incorporated by reference.

Further, the surfactant can be a pharmaceutically acceptable ionicpolymeric surfactant, a pharmaceutically acceptable nonionic polymericsurfactant, a pharmaceutically acceptable cationic polymeric surfactant,a pharmaceutically acceptable anionic polymeric surfactant, or apharmaceutically acceptable zwitterionic polymeric surfactant. Examplesof polymeric surfactants include, but are not limited to, a graftcopolymer of a poly(methyl methacrylate) backbone with multiple (atleast one) polyethylene oxide (PEO) side chain, polyhydroxystearic acid,an alkoxylated alkyl phenol formaldehyde condensate, a polyalkyleneglycol modified polyester with fatty acid hydrophobes, a polyester,semi-synthetic derivatives thereof, or combinations thereof.

Surface active agents or surfactants, are amphipathic molecules thatconsist of a non-polar hydrophobic portion, usually a straight orbranched hydrocarbon or fluorocarbon chain containing 8-18 carbon atoms,attached to a polar or ionic hydrophilic portion. The hydrophilicportion can be nonionic, ionic or zwitterionic. The hydrocarbon chaininteracts weakly with the water molecules in an aqueous environment,whereas the polar or ionic head group interacts strongly with watermolecules via dipole or ion-dipole interactions. Based on the nature ofthe hydrophilic group, surfactants are classified into anionic,cationic, zwitterionic, nonionic and polymeric surfactants.

Suitable surfactants include, but are not limited to, ethoxylatednonylphenol comprising 9 to 10 units of ethyleneglycol, ethoxylatedundecanol comprising 8 units of ethyleneglycol, polyoxyethylene (20)sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate,polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20)sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate,sorbitan monostearate, sorbitan monooleate, ethoxylated hydrogenatedricin oils, sodium laurylsulfate, a diblock copolymer of ethyleneoxydeand propyleneoxyde, Ethylene Oxide-Propylene Oxide Block Copolymers, andtetra-functional block copolymers based on ethylene oxide and propyleneoxide, Glyceryl monoesters, Glyceryl caprate, Glyceryl caprylate,Glyceryl cocate, Glyceryl erucate, Glyceryl hydroxysterate, Glycerylisostearate, Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate,Glyceryl myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate,Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thighlycolate,Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate, Glyceryldisterate, Glyceryl sesuioleate, Glyceryl stearate lactate,Polyoxyethylene cetyl/stearyl ether, Polyoxyethylene cholesterol ether,Polyoxyethylene laurate or dilaurate, Polyoxyethylene stearate ordistearate, polyoxyethylene fatty ethers, Polyoxyethylene lauryl ether,Polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, asteroid, Cholesterol, Betasitosterol, Bisabolol, fatty acid esters ofalcohols, isopropyl myristate, Aliphati-isopropyl n-butyrate, Isopropyln-hexanoate, Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecylmyristate, alkoxylated alcohols, alkoxylated acids, alkoxylated amides,alkoxylated sugar derivatives, alkoxylated derivatives of natural oilsand waxes, polyoxyethylene polyoxypropylene block copolymers,nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20 methylglucosesesquistearate, PEG40 lanolin, PEG-40 castor oil, PEG-40 hydrogenatedcastor oil, polyoxyethylene fatty ethers, glyceryl diesters,polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, andpolyoxyethylene lauryl ether, glyceryl dilaurate, glyceryl dimystate,glyceryl distearate, semi-synthetic derivatives thereof, or mixturesthereof.

Additional suitable surfactants include, but are not limited to,non-ionic lipids, such as glyceryl laurate, glyceryl myristate, glyceryldilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, andmixtures thereof.

In additional embodiments, the surfactant is a polyoxyethylene fattyether having a polyoxyethylene head group ranging from about 2 to about100 groups, or an alkoxylated alcohol having the structureR₅—(OCH₂CH₂)_(y)—OH, wherein R₅ is a branched or unbranched alkyl grouphaving from about 6 to about 22 carbon atoms and y is between about 4and about 100, and preferably, between about 10 and about 100.Preferably, the alkoxylated alcohol is the species wherein R₅ is alauryl group and y has an average value of 23.

In a different embodiment, the surfactant is an alkoxylated alcoholwhich is an ethoxylated derivative of lanolin alcohol. Preferably, theethoxylated derivative of lanolin alcohol is laneth-10, which is thepolyethylene glycol ether of lanolin alcohol with an averageethoxylation value of 10.

Nonionic surfactants include, but are not limited to, an ethoxylatedsurfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fattyacid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan esterethoxylated, a fatty amino ethoxylated, an ethylene oxide-propyleneoxide copolymer, Bis(polyethylene glycol bis[imidazoyl carbonyl]),nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij® 35,Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor®EL, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine,n-Decyl alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecylbeta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycolmonodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630, Igepal CA-630,Methyl-6-O—(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethyleneglycol monododecyl ether, N-Nonanoyl-N-methylglucamine,N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether,Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octaethylene glycolmonotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctylether, Polyethylene glycol diglycidyl ether, Polyethylene glycol etherW-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl),Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillajabark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85,Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5,Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10,Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7,Tergitol, Type NP-9, Tergitol, Tergitol, Type TMN-10, Tergitol, TypeTMN-6, Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecylether, Tetraethylene glycol monododecyl ether, Tetraethylene glycolmonotetradecyl ether, Triethylene glycol monodecyl ether, Triethyleneglycol monododecyl ether, Triethylene glycol monohexadecyl ether,Triethylene glycol monooctyl ether, Triethylene glycol monotetradecylether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, TritonGR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, TritonX-15, Triton X-151, Triton X-200, Triton X-207, Triton® X-100, Triton®X-114, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45,Triton® X-705-70, TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61,TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85, Tyloxapol, n-Undecylbeta-D-glucopyranoside, semi-synthetic derivatives thereof, orcombinations thereof.

In addition, the nonionic surfactant can be a poloxamer. Poloxamers arepolymers made of a block of polyoxyethylene, followed by a block ofpolyoxypropylene, followed by a block of polyoxyethylene. The averagenumber of units of polyoxyethylene and polyoxypropylene varies based onthe number associated with the polymer. For example, the smallestpolymer, Poloxamer 101, consists of a block with an average of 2 unitsof polyoxyethylene, a block with an average of 16 units ofpolyoxypropylene, followed by a block with an average of 2 units ofpolyoxyethylene. Poloxamers range from colorless liquids and pastes towhite solids. In cosmetics and personal care products, Poloxamers areused in the formulation of skin cleansers, bath products, shampoos, hairconditioners, mouthwashes, eye makeup remover and other skin and hairproducts. Examples of Poloxamers include, but are not limited to,Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer123, Poloxamer 124, Poloxamer 181, Poloxamer 182, Poloxamer 183,Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235,Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335,Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer407, Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.

Suitable cationic surfactants include, but are not limited to, aquarternary ammonium compound, an alkyl trimethyl ammonium chloridecompound, a dialkyl dimethyl ammonium chloride compound, a cationichalogen-containing compound, such as cetylpyridinium chloride,Benzalkonium chloride, Benzalkonium chloride,Benzyldimethylhexadecylammonium chloride,Benzyldimethyltetradecylammonium chloride, Benzyldodecyldimethylammoniumbromide, Benzyltrimethylammonium tetrachloroiodate,Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammoniumbromide, Dodecyltrimethylammonium bromide, Dodecyltrimethylammoniumbromide, Ethylhexadecyldimethylammonium bromide, Girard's reagent T,Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium bromide,N,N′,N′-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzoniumbromide, Trimethyl(tetradecyl)ammonium bromide,1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, 1-Decanaminium,N-decyl-N,N-dimethyl-, chloride, Didecyl dimethyl ammonium chloride,2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride, Alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride, Alkyl bis(2-hydroxyethyl) benzyl ammonium chloride, Alkyldemethyl benzyl ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% O₁₂), Alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% O₁₄, 40% C₁₂, 10% O₁₆), Alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C₁₄, 23% C₁₂, 20% O₁₆), Alkyl dimethyl benzylammonium chloride, Alkyl dimethyl benzyl ammonium chloride (100% O₁₄),Alkyl dimethyl benzyl ammonium chloride (100% C₁₆), Alkyl dimethylbenzyl ammonium chloride (41% C₁₄, 28% C₁₂), Alkyl dimethyl benzylammonium chloride (47% C₁₂, 18% C₁₄), Alkyl dimethyl benzyl ammoniumchloride (55% C₁₆, 20% C₁₄), Alkyl dimethyl benzyl ammonium chloride(58% C₁₄, 28% C₁₆), Alkyl dimethyl benzyl ammonium chloride (60% C₁₄,25% C₁₂), Alkyl dimethyl benzyl ammonium chloride (61% C₁₁, 23% C₁₄),Alkyl dimethyl benzyl ammonium chloride (61% C₁₂, 23% C₁₄), Alkyldimethyl benzyl ammonium chloride (65% C₁₂, 25% C₁₄), Alkyl dimethylbenzyl ammonium chloride (67% C₁₂, 24% C₁₄), Alkyl dimethyl benzylammonium chloride (67% C₁₂, 25% C₁₄), Alkyl dimethyl benzyl ammoniumchloride (90% C₁₄, 5% C₁₂), Alkyl dimethyl benzyl ammonium chloride (93%C₁₄, 4% C₁₂), Alkyl dimethyl benzyl ammonium chloride (95% C₁₆, 5% C₁₈),Alkyl dimethyl benzyl ammonium chloride, Alkyl didecyl dimethyl ammoniumchloride, Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzylammonium chloride (C₁₂₋₁₆), Alkyl dimethyl benzyl ammonium chloride(C₁₂₋₁₈), Alkyl dimethyl benzyl ammonium chloride, dialkyl dimethylbenzyl ammonium chloride, Alkyl dimethyl dimethybenzyl ammoniumchloride, Alkyl dimethyl ethyl ammonium bromide (90% C₁₄, 5% C₁₆, 5%C₁₂), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl and alkenylgroups as in the fatty acids of soybean oil), Alkyl dimethyl ethylbenzylammonium chloride, Alkyl dimethyl ethylbenzyl ammonium chloride (60%C₁₄), Alkyl dimethyl isopropylbenzyl ammonium chloride (50% C₁₂, 30%C₁₄, 17% C₁₆, 3% C₁₈), Alkyl trimethyl ammonium chloride (58% C₁₈, 40%C₁₆, 1% C₁₄, 1% C₁₂), Alkyl trimethyl ammonium chloride (90% C₁₈, 10%C₁₆), Alkyldimethyl-(ethylbenzyl) ammonium chloride (C₁₂₋₁₈),Di-(C₈₋₁₀)-alkyl dimethyl ammonium chlorides, Dialkyl dimethyl ammoniumchloride, Dialkyl methyl benzyl ammonium chloride, Didecyl dimethylammonium chloride, Diisodecyl dimethyl ammonium chloride, Dioctyldimethyl ammonium chloride, Dodecyl bis(2-hydroxyethyl) octyl hydrogenammonium chloride, Dodecyl dimethyl benzyl ammonium chloride,Dodecylcarbamoyl methyl dimethyl benzyl ammonium chloride, Heptadecylhydroxyethylimidazolinium chloride,Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine,Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium chloride(and) Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammonium chloridepolymer, n-Tetradecyl dimethyl benzyl ammonium chloride monohydrate,Octyl decyl dimethyl ammonium chloride, Octyl dodecyl dimethyl ammoniumchloride, Octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride,Oxydiethylenebis(alkyl dimethyl ammonium chloride), Quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride, Trimethoxysily propyldimethyl octadecyl ammonium chloride, Trimethoxysilyl quats, Trimethyldodecylbenzyl ammonium chloride, semi-synthetic derivatives thereof, andcombinations thereof.

Exemplary cationic halogen-containing compounds include, but are notlimited to, cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides, ortetradecyltrimethylammonium halides. In some particular embodiments,suitable cationic halogen containing compounds comprise, but are notlimited to, cetylpyridinium chloride (CPC), cetyltrimethylammoniumchloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide(CPB), cetyltrimethylammonium bromide (CTAB), cetyidimethylethylammoniumbromide, cetyltributylphosphonium bromide, dodecyltrimethylammoniumbromide, and tetrad ecyltrimethylammonium bromide. In particularlypreferred embodiments, the cationic halogen containing compound is CPC,although the compositions of the present invention are not limited toformulation with an particular cationic containing compound.

Suitable anionic surfactants include, but are not limited to, acarboxylate, a sulphate, a sulphonate, a phosphate, chenodeoxycholicacid, chenodeoxycholic acid sodium salt, cholic acid, ox or sheep bile,Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid, Deoxycholic acidmethyl ester, Digitonin, Digitoxigenin, N,N-DimethyldodecylamineN-oxide, Docusate sodium salt, Glycochenodeoxycholic acid sodium salt,Glycocholic acid hydrate, synthetic, Glycocholic acid sodium salthydrate, synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholicacid sodium salt, Glycodeoxycholic acid sodium salt, Glycolithocholicacid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester,N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine solution,N-Lauroylsarcosine solution, Lithium dodecyl sulfate, Lithium dodecylsulfate, Lithium dodecyl sulfate, Lugol solution, Niaproof 4, Type 4,1-Octanesulfonic acid sodium salt, Sodium 1-butanesulfonate, Sodium1-decanesulfonate, Sodium 1-decanesulfonate, Sodium 1-dodecanesulfonate,Sodium 1-heptanesulfonate anhydrous, Sodium 1-heptanesulfonateanhydrous, Sodium 1-nonanesulfonate, Sodium 1-propanesulfonatemonohydrate, Sodium 2-bromoethanesulfonate, Sodium cholate hydrate,Sodium choleate, Sodium deoxycholate, Sodium deoxycholate monohydrate,Sodium dodecyl sulfate, Sodium hexanesulfonate anhydrous, Sodium octylsulfate, Sodium pentanesulfonate anhydrous, Sodium taurocholate,Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid sodiumsalt monohydrate, Taurohyodeoxycholic acid sodium salt hydrate,Taurolithocholic acid 3-sulfate disodium salt, Tauroursodeoxycholic acidsodium salt, Trizma® dodecyl sulfate, TWEEN® 80, Ursodeoxycholic acid,semi-synthetic derivatives thereof, and combinations thereof.

Suitable zwitterionic surfactants include, but are not limited to, anN-alkyl betaine, lauryl amindo propyl dimethyl betaine, an alkyldimethyl glycinate, an N-alkyl amino propionate, CHAPS, minimum 98%(TLC), CHAPS, SigmaUltra, minimum 98% (TLC), CHAPS, for electrophoresis,minimum 98% (TLC), CHAPSO, minimum 98%, CHAPSO, SigmaUltra, CHAPSO, forelectrophoresis, 3-(Decyldimethylammonio)propanesulfonate inner salt,3-Dodecyldimethyl-ammonio)propanesulfonate inner salt, SigmaUltra,3-(Dodecyldimethylammonio)-propanesulfonate inner salt,3-(N,N-Dimethylmyristylammonio)propanesulfonate,3-(N,N-Dimethyloctadecylammonio)propanesulfonate,3-(N,N-Dimethyloctyl-ammonio)propanesulfonate inner salt,3-(N,N-Dimethylpalmitylammonio)-propanesulfonate, semi-syntheticderivatives thereof, and combinations thereof.

In some embodiments, the nanoemulsion RSV vaccine comprises a cationicsurfactant, which can be cetylpyridinium chloride. In other embodimentsof the invention, the nanoemulsion RSV vaccine comprises a cationicsurfactant, and the concentration of the cationic surfactant is lessthan about 5.0% and greater than about 0.001%. In yet another embodimentof the invention, the nanoemulsion RSV vaccine comprises a cationicsurfactant, and the concentration of the cationic surfactant is selectedfrom the group consisting of less than about 5%, less than about 4.5%,less than about 4.0%, less than about 3.5%, less than about 3.0%, lessthan about 2.5%, less than about 2.0%, less than about 1.5%, less thanabout 1.0%, less than about 0.90%, less than about 0.80%, less thanabout 0.70%, less than about 0.60%, less than about 0.50%, less thanabout 0.40%, less than about 0.30%, less than about 0.20%, or less thanabout 0.10%. Further, the concentration of the cationic agent in thenanoemulsion vaccine is greater than about 0.002%, greater than about0.003%, greater than about 0.004%, greater than about 0.005%, greaterthan about 0.006%, greater than about 0.007%, greater than about 0.008%,greater than about 0.009%, greater than about 0.010%, or greater thanabout 0.001%. In one embodiment, the concentration of the cationic agentin the nanoemulsion vaccine is less than about 5.0% and greater thanabout 0.001%.

In another embodiment of the invention, the nanoemulsion vaccinecomprises at least one cationic surfactant and at least one non-cationicsurfactant. The non-cationic surfactant is a nonionic surfactant, suchas a polysorbate (Tween), such as polysorbate 80 or polysorbate 20. Inone embodiment, the non-ionic surfactant is present in a concentrationof about 0.01% to about 5.0%, or the non-ionic surfactant is present ina concentration of about 0.1% to about 3%. In yet another embodiment ofthe invention, the nanoemulsion vaccine comprises a cationic surfactantpresent in a concentration of about 0.01% to about 2%, in combinationwith a nonionic surfactant.

In certain embodiments, the nanoemulsion further comprises a cationichalogen containing compound. The present invention is not limited to aparticular cationic halogen containing compound. A variety of cationichalogen containing compounds are contemplated including, but not limitedto, cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides, andtetradecyltrimethylammonium halides. The present invention nanoemulsionis also not limited to a particular halide. A variety of halides arecontemplated including, but not limited to, halide selected from thegroup consisting of chloride, fluoride, bromide, and iodide.

In still further embodiments, the nanoemulsion further comprises aquaternary ammonium containing compound. The present invention is notlimited to a particular quaternary ammonium containing compound. Avariety of quaternary ammonium containing compounds are contemplatedincluding, but not limited to, Alkyl dimethyl benzyl ammonium chloride,dialkyl dimethyl ammonium chloride, n-Alkyl dimethyl benzyl ammoniumchloride, n-Alkyl dimethyl ethylbenzyl ammonium chloride, Dialkyldimethyl ammonium chloride, and n-Alkyl dimethyl benzyl ammoniumchloride.

In one embodiment, the nanoemulsion and/or nanoemulsion vaccinecomprises a cationic surfactant which is cetylpyridinium chloride (CPC).CPC may have a concentration in the nanoemulsion RSV vaccine of lessthan about 5.0% and greater than about 0.001%, or further, may have aconcentration of less than about 5%, less than about 4.5%, less thanabout 4.0%, less than about 3.5%, less than about 3.0%, less than about2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%,less than about 0.90%, less than about 0.80%, less than about 0.70%,less than about 0.60%, less than about 0.50%, less than about 0.40%,less than about 0.30%, less than about 0.20%, less than about 0.10%,greater than about 0.001%, greater than about 0.002%, greater than about0.003%, greater than about 0.004%, greater than about 0.005%, greaterthan about 0.006%, greater than about 0.007%, greater than about 0.008%,greater than about 0.009%, and greater than about 0.010%.

In a further embodiment, the nanoemulsion RSV vaccine comprises anon-ionic surfactant, such as a polysorbate surfactant, which may bepolysorbate 80 or polysorbate 20, and may have a concentration of about0.01% to about 5.0%, or about 0.1% to about 3% of polysorbate 80. Thenanoemulsion RSV vaccine may further comprise at least one preservative.In another embodiment of the invention, the nanoemulsion RSV vaccinecomprises a chelating agent.

6. Additional Ingredients

Additional compounds suitable for use in the nanoemulsion RSV vaccinesof the invention include but are not limited to one or more solvents,such as an organic phosphate-based solvent, bulking agents, coloringagents, pharmaceutically acceptable excipients, a preservative, pHadjuster, buffer, chelating agent, etc. The additional compounds can beadmixed into a previously emulsified nanoemulsion vaccine, or theadditional compounds can be added to the original mixture to beemulsified. In certain of these embodiments, one or more additionalcompounds are admixed into an existing nanoemulsion compositionimmediately prior to its use.

Suitable preservatives in the nanoemulsion RSV vaccines of the inventioninclude, but are not limited to, cetylpyridinium chloride, benzalkoniumchloride, benzyl alcohol, chlorhexidine, imidazolidinyl urea, phenol,potassium sorbate, benzoic acid, bronopol, chlorocresol, paraben esters,phenoxyethanol, sorbic acid, alpha-tocophernol, ascorbic acid, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, sodiumascorbate, sodium metabisulphite, citric acid, edetic acid,semi-synthetic derivatives thereof, and combinations thereof. Othersuitable preservatives include, but are not limited to, benzyl alcohol,chlorhexidine (bis (p-chlorophenyldiguanido) hexane), chlorphenesin(3-(-4-chloropheoxy)-propane-1,2-diol), Kathon CG (methyl andmethylchloroisothiazolinone), parabens (methyl, ethyl, propyl, butylhydrobenzoates), phenoxyethanol (2-phenoxyethanol), sorbic acid(potassium sorbate, sorbic acid), Phenonip (phenoxyethanol, methyl,ethyl, butyl, propyl parabens), Phenoroc (phenoxyethanol 0.73%, methylparaben 0.2%, propyl paraben 0.07%), Liquipar Oil (isopropyl, isobutyl,butylparabens), Liquipar PE (70% phenoxyethanol, 30% liquipar oil),Nipaguard MPA (benzyl alcohol (70%), methyl & propyl parabens),Nipaguard MPS (propylene glycol, methyl & propyl parabens), Nipasept(methyl, ethyl and propyl parabens), Nipastat (methyl, butyl, ethyl andpropyel parabens), Elestab 388 (phenoxyethanol in propylene glycol pluschlorphenesin and methylparaben), and Killitol (7.5% chlorphenesin and7.5% methyl parabens).

The nanoemulsion RSV vaccine may further comprise at least one pHadjuster. Suitable pH adjusters in the nanoemulsion vaccine of theinvention include, but are not limited to, diethyanolamine, lactic acid,monoethanolamine, triethylanolamine, sodium hydroxide, sodium phosphate,semi-synthetic derivatives thereof, and combinations thereof.

In addition, the nanoemulsion RSV vaccine can comprise a chelatingagent. In one embodiment of the invention, the chelating agent ispresent in an amount of about 0.0005% to about 1%. Examples of chelatingagents include, but are not limited to, ethylenediamine,ethylenediaminetetraacetic acid (EDTA), phytic acid, polyphosphoricacid, citric acid, gluconic acid, acetic acid, lactic acid, anddimercaprol, and a preferred chelating agent isethylenediaminetetraacetic acid.

The nanoemulsion RSV vaccine can comprise a buffering agent, such as apharmaceutically acceptable buffering agent. Examples of bufferingagents include, but are not limited to,2-Amino-2-methyl-1,3-propanediol, ≧99.5% (NT),2-Amino-2-methyl-1-propanol, ≧99.0% (GC), L-(+)-Tartaric acid, ≧99.5%(T), ACES, ≧99.5% (T), ADA, ≧99.0% (T), Acetic acid, ≧99.5% (GC/T),Acetic acid, for luminescence, ≧99.5% (GC/T), Ammonium acetate solution,for molecular biology, ˜5 M in H₂O, Ammonium acetate, for luminescence,≧99.0% (calc. on dry substance, T), Ammonium bicarbonate, ≧99.5% (T),Ammonium citrate dibasic, ≧99.0% (T), Ammonium formate solution, 10 M inH₂O, Ammonium formate, ≧99.0% (calc. based on dry substance, NT),Ammonium oxalate monohydrate, ≧99.5% (RT), Ammonium phosphate dibasicsolution, 2.5 M in H₂O, Ammonium phosphate dibasic, ≧99.0% (T), Ammoniumphosphate monobasic solution, 2.5 M in H₂O, Ammonium phosphatemonobasic, ≧99.5% (T), Ammonium sodium phosphate dibasic tetrahydrate,≧99.5% (NT), Ammonium sulfate solution, for molecular biology, 3.2 M inH₂O, Ammonium tartrate dibasic solution, 2 M in H₂O (colorless solutionat 20° C.), Ammonium tartrate dibasic, ≧99.5% (T), BES buffered saline,for molecular biology, 2× concentrate, BES, ≧99.5% (T), BES, formolecular biology, ≧99.5% (T), BICINE buffer Solution, for molecularbiology, 1 M in H₂O, BICINE, ≧99.5% (T), BIS-TRIS, ≧99.0% (NT),Bicarbonate buffer solution, >0.1 M Na₂CO₃, >0.2 M NaHCO₃, Boric acid,≧99.5% (T), Boric acid, for molecular biology, ≧99.5% (T), CAPS, ≧99.0%(TLC), CHES, ≧99.5% (T), Calcium acetate hydrate, ≧99.0% (calc. on driedmaterial, KT), Calcium carbonate, precipitated, ≧99.0% (KT), Calciumcitrate tribasic tetrahydrate, ≧98.0% (calc. on dry substance, KT),Citrate Concentrated Solution, for molecular biology, 1 M in H₂O, Citricacid, anhydrous, ≧99.5% (T), Citric acid, for luminescence, anhydrous,≧99.5% (T), Diethanolamine, ≧99.5% (GC), EPPS, ≧99.0% (T),Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecularbiology, ≧99.0% (T), Formic acid solution, 1.0 M in H₂O, Gly-Gly-Gly,≧99.0% (NT), Gly-Gly, ≧99.5% (NT), Glycine, ≧99.0% (NT), Glycine, forluminescence, ≧99.0% (NT), Glycine, for molecular biology, ≧99.0% (NT),HEPES buffered saline, for molecular biology, 2× concentrate, HEPES,≧99.5% (T), HEPES, for molecular biology, ≧99.5% (T), Imidazole bufferSolution, 1 M in H₂O, Imidazole, ≧99.5% (GC), Imidazole, forluminescence, ≧99.5% (GC), Imidazole, for molecular biology, ≧99.5%(GC), Lipoprotein Refolding Buffer, Lithium acetate dihydrate, ≧99.0%(NT), Lithium citrate tribasic tetrahydrate, ≧99.5% (NT), MES hydrate,≧99.5% (T), MES monohydrate, for luminescence, ≧99.5% (T), MES solution,for molecular biology, 0.5 M in H₂O, MOPS, ≧99.5% (T), MOPS, forluminescence, ≧99.5% (T), MOPS, for molecular biology, ≧99.5% (T),Magnesium acetate solution, for molecular biology, ˜1 M in H₂O,Magnesium acetate tetrahydrate, ≧99.0% (KT), Magnesium citrate tribasicnonahydrate, ≧98.0% (calc. based on dry substance, KT), Magnesiumformate solution, 0.5 M in H₂O, Magnesium phosphate dibasic trihydrate,≧98.0% (KT), Neutralization solution for the in-situ hybridization forin-situ hybridization, for molecular biology, Oxalic acid dihydrate,≧99.5% (RT), PIPES, ≧99.5% (T), PIPES, for molecular biology, ≧99.5%(T), Phosphate buffered saline, solution (autoclaved), Phosphatebuffered saline, washing buffer for peroxidase conjugates in WesternBlotting, 10× concentrate, piperazine, anhydrous, ≧99.0% (T), PotassiumD-tartrate monobasic, ≧99.0% (T), Potassium acetate solution, formolecular biology, Potassium acetate solution, for molecular biology, 5M in H₂O, Potassium acetate solution, for molecular biology, ˜1 M inH₂O, Potassium acetate, ≧99.0% (NT), Potassium acetate, forluminescence, 99.0% (NT), Potassium acetate, for molecular biology,≧99.0% (NT), Potassium bicarbonate, ≧99.5% (T), Potassium carbonate,anhydrous, ≧99.0% (T), Potassium chloride, ≧99.5% (AT), Potassiumcitrate monobasic, ≧99.0% (dried material, NT), Potassium citratetribasic solution, 1 M in H₂O, Potassium formate solution, 14 M in H₂O,Potassium formate, ≧99.5% (NT), Potassium oxalate monohydrate, ≧99.0%(RT), Potassium phosphate dibasic, anhydrous, ≧99.0% (T), Potassiumphosphate dibasic, for luminescence, anhydrous, ≧99.0% (T), Potassiumphosphate dibasic, for molecular biology, anhydrous, ≧99.0% (T),Potassium phosphate monobasic, anhydrous, ≧99.5% (T), Potassiumphosphate monobasic, for molecular biology, anhydrous, ≧99.5% (T),Potassium phosphate tribasic monohydrate, ≧95% (T), Potassium phthalatemonobasic, ≧99.5% (T), Potassium sodium tartrate solution, 1.5 M in H₂O,Potassium sodium tartrate tetrahydrate, ≧99.5% (NT), Potassiumtetraborate tetrahydrate, ≧99.0% (T), Potassium tetraoxalate dihydrate,≧99.5% (RT), Propionic acid solution, 1.0 M in H₂O, STE buffer solution,for molecular biology, pH 7.8, STET buffer solution, for molecularbiology, pH 8.0, Sodium 5,5-diethylbarbiturate, ≧99.5% (NT), Sodiumacetate solution, for molecular biology,-3 M in H₂O, Sodium acetatetrihydrate, 99.5% (NT), Sodium acetate, anhydrous, ≧99.0% (NT), Sodiumacetate, for luminescence, anhydrous, ≧99.0% (NT), Sodium acetate, formolecular biology, anhydrous, ≧99.0% (NT), Sodium bicarbonate, ≧99.5%(T), Sodium bitartrate monohydrate, ≧99.0% (T), Sodium carbonatedecahydrate, ≧99.5% (T), Sodium carbonate, anhydrous, ≧99.5% (calc. ondry substance, T), Sodium citrate monobasic, anhydrous, ≧99.5% (T),Sodium citrate tribasic dihydrate, ≧99.0% (NT), Sodium citrate tribasicdihydrate, for luminescence, ≧99.0% (NT), Sodium citrate tribasicdihydrate, for molecular biology, ≧99.5% (NT), Sodium formate solution,8 M in H₂O, Sodium oxalate, ≧99.5% (RT), Sodium phosphate dibasicdihydrate, ≧99.0% (T), Sodium phosphate dibasic dihydrate, forluminescence, 99.0% (T), Sodium phosphate dibasic dihydrate, formolecular biology, ≧99.0% (T), Sodium phosphate dibasic dodecahydrate,≧99.0% (T), Sodium phosphate dibasic solution, 0.5 M in H₂O, Sodiumphosphate dibasic, anhydrous, ≧99.5% (T), Sodium phosphate dibasic, formolecular biology, ≧99.5% (T), Sodium phosphate monobasic dihydrate,≧99.0% (T), Sodium phosphate monobasic dihydrate, for molecular biology,≧99.0% (T), Sodium phosphate monobasic monohydrate, for molecularbiology, ≧99.5% (T), Sodium phosphate monobasic solution, 5 M in H₂O,Sodium pyrophosphate dibasic, ≧99.0% (T), Sodium pyrophosphatetetrabasic decahydrate, ≧99.5% (T), Sodium tartrate dibasic dihydrate,≧99.0% (NT), Sodium tartrate dibasic solution, 1.5 M in H₂O (colorlesssolution at 20° C.), Sodium tetraborate decahydrate, ≧99.5% (T), TAPS,≧99.5% (T), TES, ≧99.5% (calc. based on dry substance, T), TM buffersolution, for molecular biology, pH 7.4, TNT buffer solution, formolecular biology, pH 8.0, TRIS Glycine buffer solution, 10×concentrate, TRIS acetate—EDTA buffer solution, for molecular biology,TRIS buffered saline, 10× concentrate, TRIS glycine SDS buffer solution,for electrophoresis, 10× concentrate, TRIS phosphate-EDTA buffersolution, for molecular biology, concentrate, 10× concentrate, Tricine,≧99.5% (NT), Triethanolamine, ≧99.5% (GC), Triethylamine, 99.5% (GC),Triethylammonium acetate buffer, volatile buffer,-1.0 M in H₂O,Triethylammonium phosphate solution, volatile buffer, ˜1.0 M in H₂O,Trimethylammonium acetate solution, volatile buffer, ˜1.0 M in H₂O,Trimethylammonium phosphate solution, volatile buffer, ˜1 M in H₂O,Tris-EDTA buffer solution, for molecular biology, concentrate, 100×concentrate, Tris-EDTA buffer solution, for molecular biology, pH 7.4,Tris-EDTA buffer solution, for molecular biology, pH 8.0, Trizma®acetate, ≧99.0% (NT), Trizma® base, ≧99.8% (T), Trizma® base, ≧99.8%(T), Trizma® base, for luminescence, ≧99.8% (T), Trizma® base, formolecular biology, ≧99.8% (T), Trizma® carbonate, ≧98.5% (T), Trizma®hydrochloride buffer solution, for molecular biology, pH 7.2, Trizma®hydrochloride buffer solution, for molecular biology, pH 7.4, Trizma®hydrochloride buffer solution, for molecular biology, pH 7.6, Trizma®hydrochloride buffer solution, for molecular biology, pH 8.0, Trizma®hydrochloride, ≧99.0% (AT), Trizma® hydrochloride, for luminescence,≧99.0% (AT), Trizma® hydrochloride, for molecular biology, ≧99.0% (AT),and Trizma® maleate, ≧99.5% (NT).

The nanoemulsion RSV vaccine can comprise one or more emulsifying agentsto aid in the formation of emulsions. Emulsifying agents includecompounds that aggregate at the oil/water interface to form a kind ofcontinuous membrane that prevents direct contact between two adjacentdroplets. Certain embodiments of the present invention featurenanoemulsion vaccines that may readily be diluted with water or anotheraqueous phase to a desired concentration without impairing their desiredproperties.

7. Immune Modulators

As noted above, the RSV vaccine can further comprise one or more immunemodulators. Examples of immune modulators include, but are not limitedto, chitosan and glucan. An immune modulator can be present in thevaccine composition at any pharmaceutically acceptable amount including,but not limited to, from about 0.001% up to about 10%, and any amountinbetween, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%,about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

V. Pharmaceutical Compositions

The nanoemulsion RSV subunit vaccines of the invention may be formulatedinto pharmaceutical compositions that comprise the nanoemulsion RSVvaccine in a therapeutically effective amount and suitable,pharmaceutically-acceptable excipients for pharmaceutically acceptabledelivery. Such excipients are well known in the art.

By the phrase “therapeutically effective amount” it is meant any amountof the nanoemulsion RSV vaccine that is effective in preventing,treating or ameliorating a disease caused by the RSV pathogen associatedwith the immunogen administered in the composition comprising thenanoemulsion RSV vaccine. By “protective immune response” it is meantthat the immune response is associated with prevention, treating, oramelioration of a disease. Complete prevention is not required, thoughis encompassed by the present invention. The immune response can beevaluated using the methods discussed herein or by any method known by aperson of skill in the art.

Intranasal administration includes administration via the nose, eitherwith or without concomitant inhalation during administration. Suchadministration is typically through contact by the compositioncomprising the nanoemulsion RSV vaccine with the nasal mucosa, nasalturbinates or sinus cavity. Administration by inhalation comprisesintranasal administration, or may include oral inhalation. Suchadministration may also include contact with the oral mucosa, bronchialmucosa, and other epithelia.

Exemplary dosage forms for pharmaceutical administration are describedherein. Examples include but are not limited to liquids, ointments,creams, emulsions, lotions, gels, bioadhesive gels, sprays, aerosols,pastes, foams, sunscreens, capsules, microcapsules, suspensions,pessary, powder, semi-solid dosage form, etc.

The pharmaceutical nanoemulsion RSV vaccines may be formulated forimmediate release, sustained release, controlled release, delayedrelease, or any combinations thereof, into the epidermis or dermis. Insome embodiments, the formulations may comprise a penetration-enhancingagent. Suitable penetration-enhancing agents include, but are notlimited to, alcohols such as ethanol, triglycerides and aloecompositions. The amount of the penetration-enhancing agent may comprisefrom about 0.5% to about 40% by weight of the formulation.

The nanoemulsion RSV vaccines of the invention can be applied and/ordelivered utilizing electrophoretic delivery/electrophoresis. Further,the composition may be a transdermal delivery system such as a patch oradministered by a pressurized or pneumatic device (i.e., “gene gun”).Such methods, which comprise applying an electrical current, are wellknown in the art.

The pharmaceutical nanoemulsion RSV vaccines for administration may beapplied in a single administration or in multiple administrations.

If applied topically, the nanoemulsion RSV vaccines may be occluded orsemi-occluded. Occlusion or semi-occlusion may be performed byoverlaying a bandage, polyoleofin film, article of clothing, impermeablebarrier, or semi-impermeable barrier to the topical preparation.

An exemplary nanoemulsion adjuvant composition according to theinvention is designated “W₈₀5EC” adjuvant. The composition of W₈₀5ECadjuvant is shown in the table below (Table 1). The mean droplet sizefor the W₈₀5EC adjuvant is ˜400 nm. All of the components of thenanoemulsion are included on the FDA inactive ingredient list forApproved Drug Products.

TABLE 1 W₈₀5EC Formulation W₈₀5EC-Adjuvant Function Mean Droplet Size ≈400 nm Aqueous Diluent Purified Water, USP Hydrophobic Oil (Core)Soybean Oil, USP (super refined) Organic Solvent Dehydrated Alcohol, USP(anhydrous ethanol) Surfactant Polysorbate 80, NF Emulsifying AgentCetylpyridinium Chloride, USP Preservative

The nanoemulsion adjuvants are formed by emulsification of an oil,purified water, nonionic detergent, organic solvent and surfactant, suchas a cationic surfactant. An exemplary specific nanoemulsion adjuvant isdesignated as “60% W₈₀5EC”. The 60% W₈₀5EC-adjuvant is composed of theingredients shown in Table 2 below: purified water, USP; soybean oilUSP; Dehydrated Alcohol, USP [anhydrous ethanol]; Polysorbate 80, NF andcetylpyridinium chloride, USP (CPCAII components of this exemplarynanoemulsion are included on the FDA list of approved inactiveingredients for Approved Drug Products.

TABLE 2 Composition of 60% W₈₀5EC-Adjuvant (w/w %) Ingredients 60%W₈₀5EC Purifed Watex, USP 54.10% Soybean Oil, USP 37.67% DehydratedAlcohol, USP (anhydrous ethanol)  4.04% Polysorbate 80, NF  3.55%Cetylpyridinium Chloride, USP  0.64%

Target patient populations for treatment include, but are not limitedto, infants, elderly, transplant patients, and chronic obstructivepulmonary disease (COPD) patients.

VI. Methods of Manufacture

The nanoemulsions of the invention can be formed using classic emulsionforming techniques. See e.g., U.S. 2004/0043041. In an exemplary method,the oil is mixed with the aqueous phase under relatively high shearforces (e.g., using high hydraulic and mechanical forces) to obtain ananoemulsion comprising oil droplets having an average diameter of lessthan about 1000 nm. Some embodiments of the invention employ ananoemulsion having an oil phase comprising an alcohol such as ethanol.The oil and aqueous phases can be blended using any apparatus capable ofproducing shear forces sufficient to form an emulsion, such as FrenchPresses or high shear mixers (e.g., FDA approved high shear mixers areavailable, for example, from Admix, Inc., Manchester, N.H.). Methods ofproducing such emulsions are described in U.S. Pat. Nos. 5,103,497 and4,895,452, herein incorporated by reference in their entireties.

In an exemplary embodiment, the nanoemulsions used in the methods of theinvention comprise droplets of an oily discontinuous phase dispersed inan aqueous continuous phase, such as water or PBS. The nanoemulsions ofthe invention are stable, and do not deteriorate even after long storageperiods. Certain nanoemulsions of the invention are non-toxic and safewhen swallowed, inhaled, or contacted to the skin of a subject.

The compositions of the invention can be produced in large quantitiesand are stable for many months at a broad range of temperatures. Thenanoemulsion can have textures ranging from that of a semi-solid creamto that of a thin lotion, to that of a liquid and can be appliedtopically by any pharmaceutically acceptable method as stated above,e.g., by hand, or nasal drops/spray.

As stated above, at least a portion of the emulsion may be in the formof lipid structures including, but not limited to, unilamellar,multilamellar, and paucliamellar lipid vesicles, micelles, and lamellarphases.

The present invention contemplates that many variations of the describednanoemulsions will be useful in the methods of the present invention. Todetermine if a candidate nanoemulsion is suitable for use with thepresent invention, three criteria are analyzed. Using the methods andstandards described herein, candidate emulsions can be easily tested todetermine if they are suitable. First, the desired ingredients areprepared using the methods described herein, to determine if ananoemulsion can be formed. If a nanoemulsion cannot be formed, thecandidate is rejected. Second, the candidate nanoemulsion should form astable emulsion. A nanoemulsion is stable if it remains in emulsion formfor a sufficient period to allow its intended use. For example, fornanoemulsions that are to be stored, shipped, etc., it may be desiredthat the nanoemulsion remain in emulsion form for months to years.Typical nanoemulsions that are relatively unstable, will lose their formwithin a day. Third, the candidate nanoemulsion should have efficacy forits intended use. For example, the emulsions of the invention shouldkill or disable RSV virus to a detectable level, or induce a protectiveimmune response to a detectable level. The nanoemulsion of the inventioncan be provided in many different types of containers and deliverysystems. For example, in some embodiments of the invention, thenanoemulsions are provided in a cream or other solid or semi-solid form.The nanoemulsions of the invention may be incorporated into hydrogelformulations.

The nanoemulsions can be delivered (e.g., to a subject or customers) inany suitable container. Suitable containers can be used that provide oneor more single use or multi-use dosages of the nanoemulsion for thedesired application. In some embodiments of the invention, thenanoemulsions are provided in a suspension or liquid form. Suchnanoemulsions can be delivered in any suitable container including spraybottles and any suitable pressurized spray device. Such spray bottlesmay be suitable for delivering the nanoemulsions intranasally or viainhalation.

These nanoemulsion-containing containers can further be packaged withinstructions for use to form kits.

An exemplary method for manufacturing a vaccine according to theinvention for the treatment or prevention of RSV infection in humanscomprises: (1) synthesizing in an eukaryotic host, a full length orfragment RSV surface antigen, such as F protein; and/or (2) synthesizingin an eukaryotic host, a full length or fragment RSV surface antigen,such as G protein, wherein the synthesizing is performed utilizingrecombinant DNA genetics vectors and constructs. The one or more surfaceantigens can then be isolated from the eukaryotic host, followed byformulating the one or more RSV surface antigens with an oil in waternanoemulsion. In a further method, whole RSV virions can be cultured inan eukaryotic host, following which the RSV virions can be isolated fromthe eukaryotic host. The isolated RSV virions can then be formulatedwith the isolated surface antigens in an oil-in-water nanoemulsion. Theeukaryotic host can be, for example, a mammalian cell, a yeast cell, oran insect cell.

VII. Examples

The invention is further described by reference to the followingexamples, which are provided for illustration only. The invention is notlimited to the examples, but rather includes all variations that areevident from the teachings provided herein. All publicly availabledocuments referenced herein, including but not limited to U.S. patents,are specifically incorporated by reference.

Example 1

The purpose of this example was to describe preparation of ananoemulsion to be used in a nanoemulsion RSV vaccine.

To manufacture the nanoemulsion, the water soluble ingredients are firstdissolved in water. The soybean oil is then added and the mixture ismixed using high shear homogenization and/or microfluidization until aviscous white emulsion is formed. The emulsion may be further dilutedwith water to yield the desired concentration of emulsion or cationicsurfactant.

The nanoemulsion (NE) composition was formulated according to Table 3.

TABLE 3 Nanoemulsion composition Component Concentration v/v Water 84.7%Soybean Oil 12.6% Ethanol 1.35% Polysorbate 80 1.18% Cetylpyridiniumchloride (CPC)  0.2%

The nanoemulsion can then be combined with one or more RSV immunogens toform a nanoemulsion RSV vaccine according to the invention.

Example 2

The purpose of this example is to describe exemplary nanoemulsionsuseful as adjuvants for an RSV vaccine.

A total of 10 nanoemulsion formulations were prepared: W₈₀5EC alone, sixW₈₀5EC+Poloxamer 407 and Poloxamer 188 (P407 and P188) formulations aswell as two W₈₀5EC+Chitosan and one W₈₀5EC+Glucan formulation have beenproduced and assessed for stability over 2 weeks under acceleratedconditions at 40° C. (Table 4). All 10 nanoemulsions were stable for atleast 2 weeks at 40° C.

TABLE 4 W₈₀5EC Formulations Ratios: Method of Particle Zeta NanoemulsionCPC:Tween: Addition of Size Potential (lot) Poloxamer Poloxamer (nm)(mV) pH W₈₀5EC 1:6 — 450 60 4.9 W₈₀5EC + 3% P407 1:6 External 500 56 5.9W₈₀5EC/P407 1:5:1 Internal 391 46 5.5 W₈₀5EC/P407 1:1:5 Internal 253 365.2 W₈₀5EC/P188 1:5:1 Internal 526 54 5.1 W₈₀5EC/P188 1:3:3 Internal 41654 5.7 W₈₀5EC/P188 1:1:5 Internal 370 47 5.2 W₈₀5EC + 0.3% 1:6 External505 60 5.7 Chitosan W₈₀5EC + 0.3% 1:6 External 523 60 5.4 ChitosanW₈₀5EC + 0.03% 1:6 External 491 41 6.3 β (1,3) Glucan

The following formulations are exemplary nanoemulsions useful in the RSVvaccines of the invention: (1) Formulation 1, W₈₀5EC (NE80), comprising:(a) CPC/Tween 80 (ratio of 1:6), and (b) Particle size ˜500 nm (Table5); and Formulation 2, W₈₀P₁₈₈5EC (NE188), comprising: (a) CPC/Tween80/P188 (ratio of 1:1:5), (b) Particle size ˜300 nm (Table 6).

TABLE 5 Formulation 1 Composition of 60% W₈₀5EC adjuvant Ingredient w/w% Distilled water 54.1 CPC 0.64 Tween 80 3.55 Ethanol 4.04 Soybean oil37.7

TABLE 6 Formulation 2 Composition of 60% W₈₀P₁₈₈5EC adjuvant Ingredientw/w % Distilled water 54.1 CPC 0.64 Tween 80 0.6 Poloxamer 188 3 Ethanol4.03 Soybean oil 37.7

Example 3 Demonstration of Associated of Nanoemulsion with Viral Antigen

Materials and Methods:

Transmission Electron Micrographs and Sectioning Technique Twenty mL ofthe NE adjuvant alone or with Fluzone® was fixed with 1% (w/v) osmiumtetroxide solution. The fixed preparations were mixed with histogel in1:10 ratio to form a solid mass. The solid mixture of was sliced intothin 1 mm slices and rinsed with double distilled deionizer water. Thecross-sectioned samples were dehydrated with ascending concentrations(30%, 50%, 70%, 90%, 100%) of component A of the Durcupan® kit (Fluka,EM #14020) in double distilled deionizer water. These samples weretransferred into embedding solution (mixture of components A, B, C andD) of the Durcupan® kit. The embedded samples were sectioned to a 75 nmthickness and placed on 300 mesh carbon-coated copper grid. The sectionson the grids were stained with saturated uranyl acetate in distilled anddeionizer water (pH 7) for 10 minutes followed by lead citrate for 5minutes. The samples were viewed with a Philips CM-100 TEM equipped witha computer controlled compustage, a high resolution (2K×2K) digitalcamera and digitally imaged and captured using X-Stream imaging software(SEM Tech Solutions, Inc., North Billerica, Mass.).

Results: Electron Micrographs:

Cross sectioned TEM of 20% W₈₀5EC NE showed NE droplets with a uniforminner core material. NE vaccine containing 30 μg of HA shows discreteantigen materials/particles inside the oil core of the droplets thatrepresent the Fluzone® antigens. Since the antigen is incorporated inthe core, and is surrounded by the core material, it is protected fromstaining by the electron dense stain. This leads to a white counterstaining effect in the core. The localization of the antigen within thecore shields the antigen-sensitive protein subunits in the emulsion, andmay protect the antigen from degradation, and thus enhancing stability.There are very few Fluzone® particles outside of the NE particles thatwere stained dark in color (FIG. 1).

Example 4

The purpose of this example was to evaluate the immunogenic potential,e.g., protective immunity to RSV, of a nanoemulsion-based recombinantF-protein vaccine, comprising W₈₀5EC (adjuvant) and recombinant Fprotein, in BALB/c mice. Rationale for the example: using recombinantprotein as opposed to killed viral preparations potentially offersnumerous advantages in regards to consistency, safety, andmanufacturing.

Animals were divided randomly into three groups. Groups were immunizedon day 0 and boosted on day 28 intranasally (into nares, half volume pernare). Animals were bled prior to prime immunization and then every 2weeks throughout the duration of the study. To examine whethervaccination with NE-F protein would affect viral clearance andimmunopathology, mice were then challenged with live, infectious RSVintranasally (10⁵ PFU) 2 weeks following the boost immunization.

Test materials: (1) 60% W₈₀5EC, diluted to a final concentration of 20%.The components of W₈₀5EC are shown in Table 7 below.

TABLE 7 W₈₀5EC Formulation Function W₈₀5EC-Adjuvant; Mean Droplet Size ≈400 nm Aqueous Diluent Purified Water, USP Hydrophobic Oil (Core)Soybean Oil, USP (super refined) Organic Solvent Dehydrated Alcohol, USP(anhydrous ethanol) Surfactant Polysorbate 80, NF Emulsifying AgentCetylpyridinium Chloride, USP Preservative(2) Recombinant F-protein: (baculovirus host—Sino Biological Inc. Cat11049-V08B); (3) Phosphate Buffered Saline (sterile) 1×: Supplied byCellGro; (4) Test animal:_BALB/c mice 8-10 weeks old, females (TheJackson Laboratory).

Review of study design: Three groups of BALB/c mice were immunizedagainst F-protein as follows: (1) Prime immunization: Group I—4.45 μgF-protein+20% W₈₀5EC at the total volume 15 μl (n=8); Group II—4.45 μgF-protein at the total volume 15 μl (n=5); and Group III—PBS at thetotal volume 15 μl (n=10); and (2) Boost immunization: Group I—10 μgF-protein+20% W₈₀5EC at the total volume 15 μl (n=8); Group II—10 μgF-protein at the total volume 15 μl (n=5); and Group III—PBS at thetotal volume 15 μl (n=10).

Animals were divided randomly into three groups. Groups were immunizedon day 0 intranasally (into nares, half volume per nare). Animals werebled every 2 weeks for the duration of the experiment. The mice wereintranasally inoculated with 10⁵ PFU L19 RSV 14 days following the finalboost.

Methods: Test formulation: The vaccine mixture was formulated asfollows. First immunization: (1) 90 μl of recombinant F protein (conc.0.445 mg/ml) was mixed with 45 μl of 60% W₈₀5EC. Final concentrations: Fprotein—0.3 mg/ml; NE—20%. Volume dose—15 μl/animal. (2) 50 ul ofrecombinant F protein (conc. 0.445 mg/ml) was mixed with 25 μl of PBS1×. Final concentrations: F protein—0.3 mg/ml; NE—0%. Volume dose—15μl/animal. For the immunization boost: (1) 90 μl of recombinant Fprotein (conc. 1 mg/ml) was mixed with 45 μl of 60% W₈₀5EC. Finalconcentrations: F protein—0.67 mg/ml; NE—15%. Volume dose—15 μl/animal;and (2) 50 ul of recombinant F protein (conc. 1 mg/ml) was mixed with 25μl of PBS 1×. Final concentrations: F protein—0.67 mg/ml; NE—0%. Volumedose—15 μl/animal.

Test Methods.

Vaccination procedure: Mice were anesthetized with isoflurane andpositioned with their heads reclined about 45° then 7.5 μl vaccine wasadministered into the left nare. The animals were re-anesthetized andrestrained as above. The remaining 7.5 μl of the vaccine wasadministered into the right nare. Physical examination: Body posture,activity, and pilorection were monitored on weekly basis for eachindividual animal in the study. Bleeding: Two, 4 and 6 weeks after thefirst immunization mice were bled by saphenous phlebotomy.

Serum ELISA:

Antigen-specific IgG, IgG1, IgG2a, IgG2b, and IgE responses weremeasured by ELISA with 5 μg/ml of F-protein for plate coating.Anti-mouse IgG-alkaline phosphatase conjugated antibodies were fromJackson ImmunoResearch Laboratories Inc. (West Grove, Pa.). Alkalinephosphatase (AP) conjugated rabbit anti-mouse IgG (H&L), IgG1, IgG2a,IgG2b, IgG2c and IgE were purchased from Rockland Immunochemicals, Inc.(Gilbertsville, Pa.).

Intranasal Challenge with Live L19 RSV:

Mice were challenged with live, infectious RSV intranasally (10⁵ PFU) 2weeks post boost immunization.

Airway Hyperreactivity (AHR):

AHR was measured using a Buxco mouse plethysmograph which isspecifically designed for the low tidal volumes (Buxco). The mouse to betested was anesthetized with sodium pentobarbital and intubated viacannulation of the trachea with an 18-gauge metal tube. The mouse wassubsequently ventilated with a Harvard pump ventilator (tidal volume=0.4ml, frequency=120 breaths/min, positive end-expiratory pressure 2.5-3.0cm H2O). The plethysmograph was sealed and readings monitored bycomputer. As the box is a closed system, a change in lung volume will berepresented by a change in box pressure (Pbox) that was measured by adifferential transducer. Once baseline levels had stabilized and initialreadings were taken, a methacholine challenge was given via tail veininjection. After determining a dose-response curve (0.01-0.5 mg), anoptimal dose was chosen, 0.250 mg of methacholine. This dose was usedthroughout the rest of the experiments in this study. After themethacholine challenge, the response was monitored and the peak airwayresistance was recorded as a measure of airway hyperreactivity.

Euthanasia and Biological Material Harvest Procedure:

The mice were euthanized by isoflurane asphyxiation. Lung-associatedlymph nodes were harvested for immune response evaluation. Intranasalinoculation of mice with Line 19 RSV, leads to an infection that isassociated with a moderate form of disease phenotype, including mucushypersecretion and inflammation. The severity of this phenotype incontrol and immunized animals was assessed using histologic analysis andQPCR for viral and cytokine gene expression as well as mucus-associatedgenes Muc5ac and Gob5.

Quantitative PCR:

The smallest lung lobe was removed and homogenized in 1 ml of Trizolreagent (Invitrogen). RNA was isolated as per manufacturer's protocol,and 5 μg was reverse-transcribed to assess gene expression. Detection ofcytokine mRNA in lung samples was determined using pre-developedprimer/probe sets (Applied Biosystems) and analyzed using an ABI Prism7500 Sequence Detection System (Applied Biosystems). Transcript levelsof Muc5ac, Gob5 were determined using custom primers, as previouslydescribed [1]. Gapdh was analyzed as an internal control and geneexpression was normalized to Gapdh. Fold changes in gene expressionlevels were calculated by comparison to the gene expression inuninfected mice, which were assigned an arbitrary value of 1. RSVtranscripts were amplified using SYBR green chemistry, by adaptingpreviously published primer sets to match the sequence of Line 19:

SVG sense: 5′-CCAAACAAACCCAATAATGATTT-3′ RSVG antisense5′-GCCCAGCAGGTTGGATTGT-3′ RSVN sense: 5′-CATCTAGCAAATACACCATCCA-3′RSVN antisense: 5′-TTCTGCACATCATAATTAGGAGTATCAA-3′ RSVF sense:5′-AATGATATGCCTATAACAAATGATCAGAA-3′ RSVF antisense:5′-TGGACATGATAGAGTAACTTTGCTGTCT-3′The levels of RSV transcripts in the lungs were expressed relative tothe number of copies of GAPDH.

Plaque Assays:

Lungs of mice were excised, weighed, and homogenized in 1×EMEM (Lonza).Homogenates were clarified by centrifugation (5000×g for 10 mins),serial dilutions were made of the supernatant and added to subconfluentVero cells. After allowing the virus to adhere for one hour, thesupernatant was removed, and replaced with 0.9% methylcellulose/EMEM.Plaques were visualized on day 5 of culture by immunohistochemicaltechniques using goat anti-RSV as the primary antibody (Millipore),HRP-rabbit anti-goat antibody as the secondary, and 4-chloronapthol(Pierce) as the substrate.

Lymph Node Restimulation:

Lung associated lymph node (LALN) cell suspensions were plated induplicate at 1×10⁶ cells per well followed by restimulation with eithermedia or RSV (M01-0.5). Cells were incubated at 37° C. for 24 hours andsupernatants collected for analysis on the BioRad Bioplex 200 systemaccording to the manufacturer's protocol. Kits (BioRad) containingantibody beads to Th cytokines (IL-17, IFNγ, IL-4, IL-5, IL-13) wereused to assay for cytokine production in each of the samples.

Histology:

Right lobes of the lungs were isolated and immediately fixed in 10%neutral buffered formalin. Lung samples were subsequently processed,embedded in paraffin, sectioned, and placed on L-lysine-coated slides,and stained using standard histological techniques using Hemotoxylin andEosin (H&E) and Periodic-acid Schiff (PAS). PAS staining was done toidentify mucus and mucus-producing cells.

Results.

Evaluation of humoral response. Evaluation of specific serum IgG. Seraobtained from mice 2, 4 and 6 weeks after the prime immunization wereused to assess the endpoint titer of specific IgG using ELISA. Endpointtiter was defined as the highest sera dilution yielding absorbance threetimes above the background. Endpoint titer results are shown in FIG. 2.Only nanoemulsoin F-protein immunized mice responded vaccination by hightiters of specific anti-F-protein IgG antibodies with group averagetiters approaching 5×10⁶ at week 6.

Evaluation of Specific IgG1, IgG2a, IgG2b, and IgE humoral response insera to immunization. Sera obtained from mice two weeks after the secondimmunization (week 6) were used to assess the endpoint titer of specificIgG1, IgG2a, IgG2b, and IgE using ELISA (FIG. 3). Endpoint titer wasdefined as the highest serum dilution yielding absorbance three timesabove the background. NE+F-protein immunized mice produced high levelsof levels of specific IgG1, IgG2a, IgG2b antibodies (FIGS. 3A, 3B and3C). Serum IgE titers were low but present and averaged around 663 forNE+F-protein immunized mice (FIG. 3D).

RSV Challenge:

RSV Gene expression in lungs 8 days following challenge. A challengestudy was conducted to determine whether vaccination with NE-F-proteinwould protect the mice from respiratory challenge with RSV. At 6 weeksfollowing prime immunization, mice were challenged with live, infectiousRSV intranasally (10⁵ PFU). On day 8 post-challenge, viral load wasassessed in the lungs via QPCR and via plaque assay. As assessed viaQPCR, a significant decrease in the transcript levels for RSV F and RSVN and RSV G were detected in the lungs of NE-F-protein vaccinated micein comparison to non-immunized and F-protein only immunized mice (FIG.4). These data indicate that NE-F-protein vaccine dramatically improvesviral clearance in the following lower respiratory challenge.

Nanoemulsion+-RSV does not promote airway hyperreactivity. As previouslyreported, vaccination with formalin fixed RSV promotes the developmentof airway hyperreactivity (AHR) and eosinophilia upon live viralchallenge. With this in mind, whether nanoemulsion+F-protein vaccinationpromotes airway hyper-reactivity, or other evidence ofimmunopotentiation, was evaluated. Compared to control RSV infectedmice, nanoemulsion-RSV immunized mice exhibited only baseline increasesin airway resistance following intravenous methacholine challenge (FIG.5).

Nanoemulsion+F-protein immunization is associated with mucus secretionfollowing live challenge. Intranasal inoculation of mice with Line 19RSV, leads to an infection that is associated with a moderate form ofdisease phenotype, including mucus hypersecretion and inflammation. Theseverity of this phenotype in control and immunized animals was assessedusing histologic analysis and QPCR for viral and cytokine geneexpression. At day 8, post-challenge, NE+F-protein vaccinated miceexhibited similar mucus hypersecretion compared to challengednon-immunized mice, as assessed via histologic analysis (FIG. 6A).Similar expression of the mucus-associated genes Muc5ac and Gob5 wasmeasured in NE-F-protein immunized mice compared to non-immunizedcontrols (FIG. 6B).

Nanoemulsion+F-protein immunization promotes induction of mixed Th1 andTh2 cytokines following challenge. The further characterize theimmunization phenotype that promoted viral clearance innanoemulsion+F-protein immunized; we used QPCR for cytokine geneexpression. Compared to control mice, nanoemulsion+F-protein vaccinatedmice did not exhibit IL-12 and IL-17, as assessed by the levels of RSVtranscripts in the lungs at day 8 post challenge (FIGS. 7A and Brespectively). As a means of validation, cytokine profiles were assessedin bronchoalveolar lavage and lung homogenates via multiplexantibody-based assay (Bioplex). NE-RSV vaccination showed an enhancedIFN-γ response. IL-17 showed increase production compared tounvaccinated mice (FIG. 7C).

Conclusions: Only the group immunized with 20% W₈₀5EC mixed withF-protein responded to vaccination with high titers of specific anti-RSVIgG, IgG1, IgG2a and IgG2b antibodies. This was associated with minimalproduction of IgE. Nanoemulsion+F-protein vaccination was alsoassociated with enhanced viral clearance and protection following liveRSV challenge. Interestingly, the phenotype of the immune response wasnot associated with production of IL-12 or IL-17.

A mixed Th1, Th2 pattern of cytokine release was observed forNE+F-protein immunized mice both in lymph nodes after re-stimulation invitro with RSV L19. However, this was not associated withimmunopotentiation although significant mucus production was observed.

Example 5

RSV is a leading cause of severe lower respiratory tract disease ininfants, elderly, and immunocompromised individuals. Currently there isno vaccine available. Antibodies against surface protein F areconsidered important in host defense against RSV infection, however,protection is incomplete and of limited duration.

Materials and Methods:

L19 RSV virus was inactivated by formulation with W₈₀5EC nanoemulsion.BALB/c mice were vaccinated intranasally at weeks 0 and 4 with 12 μL ofinactivated L19 virus containing 1.2 μg of RSV F protein at 1.3×10⁵PFU/dose+20% W₈₀5EC or recombinant RSV F at 2.5 μg/dose+20% W₈₀5EC. Bothvaccine formulations were compared to unimmunized animals uponchallenge. Serum from immunized animals was collected prior immunizationand at week 4 post second immunization. Animals were challengedoropharyngeally with 10⁵ PFU of L19 RSV strain at 10 weekspost-immunization and tested for viral RNA clearance using PCR andhistopathological change by microscopy.

Results:

Both inactivated whole virus and recombinant F protein produced animmune response and reduced viral mRNA after challenge (p<0.01 by MannWhitney). Anti-F ELISA units reached a geometric mean (GM) of 51 (95% CI14-189) following whole virus vaccination and were lower compared to aGM of 470 (95% CI-235-942) following F protein vaccination (p=0.02 byMann-Whitney). See FIG. 8. F protein was undetectable after challenge in100% of mice vaccinated with whole virus, however, whereas 100% micevaccinated with recombinant F protein had detectable F protein mRNA inlungs post challenge (p=0.008 by Fisher's exact test). See FIG. 9 andTable 8. Additionally, the histopathology of animals vaccinated withwhole virus had less mucus than animals vaccinated with F protein. SeeFIGS. 10A-10D.

TABLE 8 Number of mice with detectable RSV proteins by PCR ImmunizationGroup F Protein G Protein N Protein No immunization 5/5 5/5 5/5 FProtein Only 5/5 (0.03) 5/5 (0.010) 5/5 (2.3) RSV 0/5 3/5 0/5 RSV + Fprotein 2/5 4/5 0/5

Conclusions:

Whole virus and recombinant F protein induce an immune response andreduce viral mRNA after challenge. Despite lower antibody titer, wholevirus vaccine inactivated and adjuvanted with W₈₀5EC nanoemulsionresults in improved viral clearance and reduced histopathology uponchallenge.

Example 6

The purpose of this example is to compare HRSV Protein Expression forRSV A2 strain as compared to RSV L19 strain, and Cell Lysate vs.Supernatant.

Materials and Methods: All samples were prepared by infecting HEP-2cells with the same amount of pfu from either A2 or L19 viruses. Twentyfour hours post infection; the infected cells were treated with eitherone of the following:

(1) Cell lysate to check for the cell associated proteins; afterdiscarding the supernatant media, the cells were treated with SDS. Thiscell lysate was assayed for quantity of F protein associated with thecells.

(2) Total cell and supernatant proteins; the cells and supernatant werefrozen and thawed 3 times to lyse the cells and all the cell lysate wasused to assay the F protein in the cells and the media.

L19 and A2 virus was extracted and purified from HEP-2 infected cells 4days following infection. Purified virus was compared for proteincontents.

Results: Normalized samples were assayed in Western blots using apolyclonal anti RSV antibodies. F and G protein contents were comparedbetween L19 and A2 strains. The density of the bands was compared usingimage capturing and a Kodak software. A mock non-infected cell culturewas prepared as a control.

The results data are detailed in FIGS. 11-13 and Tables 9-11. FIG. 11shows an SDS PAGE of HRSV Infected Cell Lysate (SDS treated) with L19and A2, FIG. 12 shows an SDS-PAGE of L19 and A2 HRSV Cell Lysate (cells& supernatant), and FIG. 13 shows an SDS PAGE of HRSV L19 and A2Purified Virus. Table 9 shows comparable HRSV F and G protein from L19and A2 levels from SDS-PAGE. Table 10 shows comparable HRSV L19 and A2 Fand G protein from infected cells (Lysate, Supernatant). Finally, Table11 shows comparable HRSV L19 and A2 F and G protein from SDS PAGE.

TABLE 9 Comparable HRSV F and G Protein from L19 and A2 Levels fromSDS-PAGE Infected Infected Band Density Mock with A2 with L19 Ratio(L19/A2) G (90 kDa) No Band 78241.1 356946.3 4.56 F2 (44-45 kDa) No Band38612 121328 3.14

TABLE 10 Comparable HRSV L19 and A2 F and G Protein from Infected Cells(Lysate, Supernatant) Supernatant Lysate Infected Infected InfectedInfected with Mock with A2 with L19 Mock with A2 L19 G No Band 27831166308 0 4686 54142 (90 kDa) Ratio of 6 11.6 L19:A2 F2 No Band 1064543570 No 1860 18499 (44-45 kDa) Band Ratio of 4.1 9.9 L19:A2

TABLE 11 Comparable HRSV L19 and A2 F and G Protein From SDS PAGE A2 L19(2.3 × 10⁶ pfu) (2 × 10⁶ pfu) Ratio (L19/A2) G 5,039.1 11,401.1 2.26F0 + F2 4,481.81 9,700.39 2.16

Summary: RSV L19 virus infected cells produce between 3-11 fold higherquantities of RSV viral proteins as compared to A2 infected cells.

Example 7

The purpose of this example was to compare F protein expression in Hep-2cells infected with different strains of RSV virus (L19 vs. A2) forvarious infection times (24 hours vs. 4 days).

Materials and Methods: Hep-2 cells were infected with either L19 or A2RSV virus. 2 sets of 4 flasks total.

24 hours after virus infection, the first set of Hep-2 cells were lysedwith or without culture supernatant. Samples were prepared as thefollowing:

TABLE 12 Plate 1 Plate 2 Plate 3 Plate 4 Infect with L19 Infect with L19Infect with A2 Infect with A2 24 hrs later Discard Medium Leave mediumin Discard medium Leave medium in Add Tris Buffer Add Tris buffer (samevolume) Tris (Same volume) Lyze cells Lyze cells Lyze cells Lyze cellsLot # 1123, C + T Lot # 1124, C + M Lot # 1125, C + T Lot # 1126, C + MC + TCCC + T = Cell + Tris Buffer (culture medium was discarded andreplaced with equal volume of Tris buffer); C + M = Cell + CultureMedium (culture medium reserved).

Four days after infection, the second set of Hep-2 cells were lysed withor without culture supernatant. Samples were prepared as the following:

TABLE 13 Plate A Plate B Plate C Plate D Infected with L19 Infected withA2 4 days later Remove Medium Leave Remove Medium Leave and Save Mediumin and Save Medium in Add Saved Add buffer Saved Buffer Medium (SameMedium (same volume) volume) Lyse cells Lyse cells Lyse cells Lyse cellsC + T = Cell + Tris Buffer (culture medium was replaced with equalvolume of Tris buffer) M = Culture Medium (culture medium was collectedseparately) C + M = Cell + Culture Medium (culture medium reserved)

Some 7.5 μL of each sample was applied for Western blot analysis. Thedensity of F and G protein bands were measured using CarestreamMolecular Imaging Software 5.X.

Results: The results are detailed in FIG. 14, which shows a Western blotof HRSV L19 and A2 F and G protein expression 24 hours after virusinfection. In addition, Table 14 below shows a density analysis of HRSVF and G protein band from Western Blot.

TABLE 14 HRSV F and G Protein Band Density Analysis From Western BlotSample Collection Time 24 hours After Infection 4 Days After InfectionViraSource Sample ID 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132Virus Strain L19 A2 L19 A2 Sample Description C + T C + M C + T C + MC + T M C + M C + T M C + M Band Density G 37130.4 39563.9 5076.615489.7 70377.4 70980.1 89469.8 5986.2 18172.8 19615.9    (90 kDa) F224309.2 22565.8 2160.4 7173.5 34428.1 25094.9 41726.3 6994.2 9542.67122.8 (44-45 kDa) Concentration G 7.7 8.2 1.1 3.2 8.9 9.0 11.4 0.8 2.32.5 (μg/mL)    (90 kDa) F2 36.8 34.1 3.3 10.9 32.5 23.7 39.4 6.6 9.0 6.7(44-45 kDa)

Summary: Both cell-associated viral particles and culturemedia-associated viral particles express much higher F (about 6 foldaverage) in L19 infected cells as compared to those infected with RSV A2strain.

In addition, both cell-associated viral particles and culturemedia-associated viral particles express much higher G in L19 infectedcells compared to those infected with RSV A2 strain.

Example 8

The purpose of this example was to compare several different approachesfor inactivation of RSV, including 6-propiolactone and W₈₀5ECNanoemulsion, via nasal vaccination in a mouse.

Methods: W₈₀5EC, an oil-in-water nanoemulsion with both antiviral andadjuvant activity, was compared with 6-propiolactone (β-PL) inactivatedvirus (strain L19 @2×105 pfu/dose). The two vaccines were administeredintranasally (IN) to BALB/C mice at weeks 0 and 4. Mice were bled priorto dosing and at 3 weeks post-boost and then tested for specificantibodies against F-protein.

Animals were challenged nasally with 1×10⁵ pfu RSV L19 at week 8 andchecked for airway hyper-reactivity (AHR), lung cytokines, and viralprotein mRNA clearance using PCR.

Results: Both W₈₀5EC and β-PL completely inactivated RSV and induced animmune response. β-PL vaccine induced higher antibody response comparedto nanoemulsion-inactivated vaccine (p=0.006). Animals vaccinated withnanoemulsion-inactivated vaccine, however, had higher clearance of theRSV following the challenge, evidenced by lower proteins F and G mRNA inthe lungs (p=0.06 and 0.0004, respectively). Moreover, animals receivingnanoemulsion-inactivated vaccine demonstrated a significant lower AHR(p=0.02). Both vaccines induced significant levels of lung IL-17 ascompared to nonvaccinated control (<0.01), however, significantly higherlevels were induced by nanoemulsion-inactivated vaccine (p=0.009).

Conclusions: β-PL inactivated RSV virus vaccine is associated with AHRfollowing viral challenge in a mouse model of RSV infection. Incontrast, nanoemulsion viral inactivation produced no AHR and induced asignificantly increased IL-17 production and improved viral clearance.This suggests a novel pathway of immune protection that may providebenefit for vaccination against RSV.

Example 9

The purpose of this example is to describe RSV viral strains useful inthe vaccines of the invention.

L19 RSV strain was evaluated as an antigen in the nanoemulsioninactivated/nanoemulsion adjuvanted RSV vaccine. This strain was foundto cause infection and enhanced respiratory disease (ERD) in mice.Moreover, data published showed that it conferred protection withoutinduction of ERD in mice when formulated with nanoemulsion. This L19strain was compared to a Wildtype A2 strain obtained from the AmericanType Culture Collection (ATCC).

The RSV Strain L19 isolate was isolated from an RSV-infected infant withrespiratory illness in Ann Arbor, Mich. on 3 Jan. 1967 in WI-38 cellsand passaged in SPAFAS primary chick kidney cells followed by passage inSPAFAS primary chick lung cells prior to transfer to MRC-5 cells(Herlocher 1999) and subsequently Hep2 cells (Lukacs 2006). Comparisonof RSV L19 genome (15,191-nt; GenBank accession number FJ614813) withthe RSV strain A2 (15,222-nt; GenBank accession number M74568) showsthat 98% of the genomes are identical. Most coding differences betweenL19 and A2 are in the F and G genes. Amino acid alignment of the twostrains showed that F protein has 14 (97% identical) and G protein has20 (93% identical) amino acid differences.

RSV L19 strain has been demonstrated in animal models to mimic humaninfection by stimulating mucus production and significant induction ofIL-13 using an inoculum of 1×10⁵ plaque forming units (PFU)/mouse byintra-tracheal administration (Lukacs 2006).

Rationale for Selection of RSV L19 Strain: Importantly and uniquely, theRSV L19 viral strain is unique in that it produces significantly higheryields of F protein (approximately 10-30 fold more per PFU) than theother strains. F protein content may be a key factor in immunogenicityand the L19 strain currently elicits the most robust immune response.The L19 strain has a shorter propagation time and therefore will be moreefficient from a manufacturing perspective. NanoBio proposes to produceRSV L19 strain virus for the vaccine in a qualified Vero cell linefollowing single plaque isolation of the L19 strain and purification ofthe virus to establish a Master Viral Seed Bank and Working Viral SeedBank. The results comparing the three viral strains are provided inTable 15.

TABLE 15 Comparison of RSV Strains Days of RSV RSV Viral Propa- Fprotein G protein G/F Titer RSV Strain gation (μg/mL) (μg/mL) Ratio(PFU/mL) L19 4-5 110 603 5.5 0.5 × 10⁷ A2 Wild Type¹ 4-5 44 108 2.5 1.9× 10⁷ rA2cp248/404² 8-9 38 284 7.5 0.5 × 10⁸ ¹ATCC (strain numberVR-1540). Virus was isolated from an RSV infected infant withrespiratory illness in Melbourne, Australia in 1961 and has beenpropagated in HEp-2 cell culture at least 27 times (Lewis 1961). Thisvirus has been treated to remove adenovirus from the original depositand has been utilized as a challenge strain in human clinical trials(Lee 2004). ²Recombinant temperature-sensitive A2 mutant virus obtainedfrom the NIH (Whitehead 1998).

Example 10

The purpose of this example is to describe Inactivation of RSV L19 viralstrain with different nanoemulsion adjuvants.

The nanoemulsions (1) W₈₀5EC, (2) W₈₀5EC with P407; (3) W₈₀5EC withP188, (4) W₈₀5EC with high and low molecular weight Chitosan, and (5)W₈₀5EC with Glucan, have been tested with the RSV L19 viral strain todetermine viral inactivation.

Inactivation with 20% nanoemulsion was performed for 2 hours at roomtemperature and with 0.25% βPL for 16 hours at 4° C. followed by 2 hoursat 37° C. The treated virus was passaged three times in Hep-2 cells andWestern blot analysis was performed on cell lysate to determine presenceof live virus. See FIG. 15. In particular, FIG. 15 shows the viralinactivation by Western blot assessment, with lanes containing: (1)W₈₀5EC (Lane 1), (2) W₈₀5EC+0.03% B 1,3 Glucan (lane 2), (3) W₈₀5EC+0.3%Chitosan (medium molecular weight)+acetic acid (lane 3), (4) W₈₀5EC+0.3%P407 (lane 4), (5) W₈₀5EC+0.3% Chitosan (low molecular weight)+0.1%acetic acid (lane 5), (6) media alone (lane 6); (7) βPL-inactivatedvirus (lane 7), and (8) L19 positive control (lane 8).

RSV L19 was completely inactivated by the nanoemulsion formulationsevaluated and by βPL. FIG. 15 shows that three consecutive passages ofthe NE-treated virus in a cell culture resulted in no detected viralantigen when blotted against RSV antibodies in a western blot. Thisthree cell culture passage test is well established and accepted methodfor determining viral inactivation. Of note, all lanes in FIG. 15 have athick background band, which is not a viral band, but is bovine serumalbumin. Viral proteins can be detected only in the positive control(lane 8).

Example 11

The purpose of this example was to evaluate the short term stability ofRSV vaccines.

Target doses of RSV L19 viral preparations were formulated to achieve afinal nanoemulsion concentration of 20%. Vaccine was stored at roomtemperature (RT) and at 4° C. Stability test parameters includedphysical and chemical analysis (Table 16).

TABLE 16 Stability Test Parameters Stability Test Acceptance CriteriaPhysical Appearance No separation Mean Particle Size ±10% of initialsize Zeta Potential ±10% of initial charge Western Blot No change in Gband intensity

Physical appearance, mean particle size, zeta potential and Western Blotacceptance criteria with RSV strain L19 were met following 14 days ofstorage (longest tested) at RT and 4° C. with W₈₀5EC+/−βPL inactivation.W₈₀5EC+3% P407, W₈₀5EC+0.3% Chitosan-LMW, and W₈₀5EC+0.3% Chitosan-MMWwere tested for a maximum of 7-8 days and also demonstrated stability.The W₈₀5EC/P188 (1:1:5) and W₈₀5EC/P188 (1:5:1) formulations were alsotested with a live virus RSV A2 strain as opposed to RSV L19 strain fora maximum of 14 days; the 1:1:5 formulation demonstrated stabilitywhereas the 1:5:1 formulation demonstrated potential agglomeration(Table 17).

TABLE 17 Vaccine Stability by Physical and Chemical Parameters andWestern Blot Starting Adjuvant Zeta Composition Z-average PotentialStability Based on Viral Strain (60%) Condition (nm) # of peaks PDI (mV)G Protein pass/fail βPL inactivated Reference Fresh 542.1 2 0.199 41.5NA L19 W₈₀5EC (1:6) 4° C.-14 d 548.6 2 0.241 43.5 Pass RT-14 d 538.6 20.210 40.7 Pass L19 Reference Fresh 588.5 2 0.234 39.3 NA W₈₀5EC (1:6)4° C.-14 d 545.9 2 0.210 39.9 Pass RT-14 d 535.6 2 0.234 41.1 Pass L19 +PEG W₈₀5EC + 3% P407 Fresh 779.3 1 0.351 20.1 NA (external addition) 4°C.-8 d 654.8 1 0.313 30.4 Pass RT-8 d 763.2 1 0.313 30.2 Pass L19 + PEGW₈₀5EC + 0.3% Chitosan- Fresh 557.2 1 0.253 60.1 NA LMW 4° C.-7 d 534.71 0.234 NA Pass External Addition RT-7 d 534.7 1 0.229 62.4 Pass L19 +PEG W₈₀5EC + 0.3% Chitosan- Fresh 528.4 1 0.226 NA NA MMW 4 C-7 d 532.01 0.229 63.5 Pass External Addition RT-7 d 568.0 1 0.254 64.9 Pass A2W₈₀5EC /P188 Fresh 229.5 1 0.108 27.0 NA (1:1:5) 4° C.-14 d 259.0 20.206 27.0 Pass RT-14 d 249.9 2 0.161 20.4 Pass A2 W₈₀5EC /P188 Fresh396.1 2 0.164 37.1 NA (1:5:1) 4° C.-14 d 5544.0* 2 0.619  −4.3* PassRT-14 d 2010.0* 2 0.753 −17.1* Pass *potential agglomeration

FIG. 16 shows an example of G band intensity of RSV strain L19 withW₈₀5EC+/−βPL inactivation by Western blot at day 0 (FIG. 16A) andfollowing 14 days of storage at RT or 4° C. (FIG. 16B). In particular,FIG. 16 shows a Western blot analysis performed with anti-RSV antibody(anti-G); L19 virus 4×10⁶ PFU/lane, 2×10⁶ PFU/lane, and 1×10⁶PFU/lane+/−βPL inactivation combined with W₈₀5EC as indicated. Specimenswere analyzed fresh (FIG. 16A) or after 14 days at 4° C. or roomtemperature (RT) (FIG. 16B).

Example 12

The purpose of this example was to evaluate the immunogenicity of an RSVvaccine in mice.

Mice were immunized intramuscularly as shown in Table 18. Mice received50 μl of RSV adjuvanted vaccine IM at 0 weeks. Mice were bled on 0 and 3weeks and tested for serum antibodies. Chitosan was used as animmune-modulator to enhance the immune response in addition to theadjuvant activity contributed to the nanoemulsion.

TABLE 18 Different arms used in vaccination of the mice Arm Virus NE #of # Preparation formulation animals 1 RSV L19 − 2 μg F 2.5% W₈₀5EC +0.1% Low 10 Mol. Wt. Chitosan 2 RSV L19 − 2 μg F 5% W₈₀5EC 10 3 RSV L19− 2 μg F 2.5% W₈₀5EC 10 4 RSV L19 − 2 μg F None 10 βPL inactivated 5Naive—No 10 vaccine

Mice vaccinated with nanoemulsion containing chitosan showed moreenhanced immune response following a single dose of nanoemulsionadjuvanted RSV vaccine when compared to nanoemulsion without chitosan(FIG. 17). In particular, FIG. 17 shows the immune response (IgG, μg/ml)at week 3 following vaccination in mice vaccinated IM with differentnanoemulsion formulations with and without chitosan: (1) RSV strainL19+2.5% W₈₀5EC+0.1% Low Mol. Wt. Chitosan; (2) RSV strain L19+5%W₈₀5EC; (3) RSV strain L19+2.5% W₈₀5EC; (4) RSV strain L19+βPLinactivated virus; and (5) naive mice (no vaccine). The results depictedin FIG. 17 show the highest levels of IgG were found in mice vaccinatedwith RSV strain L19+2.5% W₈₀5EC+0.1% Low Mol. Wt. Chitosan, with thenext highest levels of IgG found in mice vaccinated with RSV strainL19+2.5% W₈₀5EC, followed by mice vaccinated with RSV strain L19+5%W₈₀5EC. The lowest levels of IgG observed in vaccinated mice were forRSV strain L19+βPL inactivated virus.

Example 13

The purpose of this example was to determine the immunogenicity of RSVvaccines according to the invention in Cotton rats.

Cotton rats are the accepted animal species for evaluatingimmunogenicity and efficacy of RSV vaccines. Using data generated inmice, two nanoemulsions were selected for evaluation in Cotton rats. Thetwo initial formulations studied include the W₈₀5EC and the W₈₀P₁₈₈5EC(1:1:5) (see Tables 5 and 6 above).

Cotton rats received two doses of 30 μl IN of thenanoemulsion-adjuvanted vaccine containing 6.6 μg F-ptn. They werechallenged with 5×10⁵ pfu RSV strain A2 at week 23. Half of the animalswere sacrificed at day 4 and half were sacrificed on day 8. Thevaccination schedule is demonstrated in FIG. 18.

Immunogenicity data presented below show that upon IN immunization withan RSV-nanoemulsion vaccine, a positive immune response was observed.Upon the administration of the second dose, a rapid and significantincrease in antibody titers were achieved. Data presented in FIG. 19show that at week 21, the antibody level in all animals was about onetenth of the maximal values obtained shortly after the administration ofthe first boost at week 4. Administration of a second boost prior to thechallenge yielded an immune response that was almost identical to levelsachieved at week 6, two weeks after the first boost. Both nanoemulsionswere equally efficient in eliciting a strong and significant immuneresponses (FIGS. 19 and 20). (The Y axis in FIGS. 19 and 20 shows theend point titers or antibody quantity of specific antibody to F proteinand the X axis shows the time period in weeks.)

ELISA Unit/μg/ml:

The amount of specific antibody to F protein was calculated by areaunder the curve in the ELISA in relation to a defined reference serumwhich was assigned an arbitrary 100 EU.

Example 14

The purpose of this example was to determine the effect of RSV vaccinesaccording to the invention on neutralizing antibodies, as well ascross-reactivity of an RSV vaccine comprising RSV strain L19 againstother RSV strains following IN administration.

Cotton rats were vaccinated with 30 μl of vaccine intranasally, boostedat 4 weeks, and bled at 0, 4, 6, and 8 weeks. Animals were challenged atweek 23 with 5×10⁵ pfu of RSV strain A2. Study groups included twogroups that received 20% W₈₀5EC nanoemulsion mixed with either 1.6×10⁵PFU RSV strain L19 containing 3.3 μg F protein (n=8) or 3.2×10⁵ PFU RSVstrain L19 containing 6.6 μg F protein (n=8), as well as two groups thatreceived 20% W₈₀P₁₈₈5EC nanoemulsion mixed with either 1.6×10⁵ PFU RSVstrain L19 containing 3.3 μg F protein (n=8) or 3.2×10⁵ PFU L19 RSVcontaining 6.6 μg F protein (n=8).

Half of the animals were sacrificed at Day 4 and half at Day 8.Individual cotton rats sera was tested for neutralizing antibodies.Neutralization units (NEU) represent a reciprocal of the highestdilution that resulted in 50% plaque reduction. NEU measurements wereperformed at 4 weeks (pre boost) and at 6 weeks (2 weeks post boost).Specimens obtained at 6 weeks generated humoral immune responsesadequate to allow for NEU analysis. Data is presented as geometric meanwith 95% confidence interval (CI) (FIG. 21A). Correlation between EU andNEU is for all animals at 6 weeks using Spearman rho (FIG. 21B).

Specifically, FIG. 21 shows neutralizing antibody titers at 6 weeks timepoint (FIG. 21A). It is noteworthy that all animals vaccinated with3.2×10⁶ PFU RSV strain L19 inactivated with 60% W₈₀5EC or 60% W₈₀P₁₈₈5ECgenerated robust neutralizing antibodies. There is a statisticallysignificant positive correlation between EU and neutralizing antibodies(NEU) (FIG. 21B).

Neutralization Unit (NU):

The reciprocal of the highest serum dilution that reduces viral plaquesby 50%.

Specific Activity (NU/EU):

Viral neutralizing antibody antibodies (NU) per the one EU F-proteinantibody (FIG. 21B)

FIG. 22 shows neutralizing antibodies on day 4 and day 8. FIG. 22A showsthe results for W₈₀P₁₈₈5EC nanoemulsion combined with RSV strain L19,and FIG. 22B shows the results for W₈₀5EC nanoemulsion combined with RSVstrain L19. All cotton rats demonstrated high neutralizing antibodies(NU) against the vaccine RSV strain L19. Neutralizing antibodies wererising steadily following the challenge (Y axis). Day 8 neutralizingunits (NU) were higher than Day 4 NU. Naïve Cotton Rats did not show anyneutralization activity in their sera. Serum neutralizing antibodies andspecific activity showed a trend to increase from Day 4 to Day 8post-challenge.

FIG. 23 shows the specific activity of serum antibodies. The specificactivity (Neutralizing units/ELISA units) of the serum antibodies tendsto increase on Day 8 when compared to Day 4 post-challenge. FIG. 23Ashows the results for W₈₀P₁₈₈5EC nanoemulsion combined with RSV strainL19 (NU/EU for the Y axis), at Day 4 and Day 8. FIG. 23B shows theresults for W₈₀5EC nanoemulsion combined with RSV strain L19 (NU/EU forthe Y axis), at Day 4 and Day 8. All cotton rats demonstrated highneutralization activity (FIG. 23).

Serum of vaccinated cotton rats showed cross protection against RSVstrain A2 (in addition to RSV strain L19) on Day 4 post-challenge (FIG.24). Specifically, FIG. 24 shows cross protection at Day 4 for cottonrats that received 3 doses of RSV L19 adjuvanted vaccine, thenchallenged with RSV strain A2. FIG. 24A shows the results for W₈₀P₁₈₈5ECnanoemulsion combined with RSV strain L19, and FIG. 24B shows theresults for W₈₀5EC nanoemulsion combined with RSV strain L19. Serumneutralization activity shows equivalent NU against RSV strain L19 orRSV strain A2, demonstrating cross protection between the two RSVstrains. Vaccinated cotton rats cleared all challenged RSV virus on Day4 post challenge when compared with naïve cotton rats (FIG. 25). Asexpected by day 8 all animals had cleared the virus. Specifically, FIG.25 shows viral clearance at Day 4 in the lungs of the cotton rats, bymeasurement of the RSV strain A2 viral titer (PFU/g) in the lungs of thetested cotton rats. Vaccinated cotton rats (vaccinated with W₈₀P₁₈₈5ECnanoemulsion combined with RSV strain L19, and W₈₀5EC nanoemulsioncombined with RSV strain L19), showed complete clearance of RSV strainA2 challenged virus from the lungs of the cotton rats. In contrast,naïve animals shows >10³ pfu RSV strain A2/gram of lung (limit ofdetection was 2.1×10¹ pfu/g).

Example 15

The purpose of this example was to evaluate Intramuscular vaccination ofRSV vaccines according to the invention in Cotton Rats.

Cotton rats were vaccinated IM according to the schedule shown in FIG.26. Animals received 50 μl RSV adjuvanted RSV vaccine containing 3.3 μgF-protein (20% W₈₀5EC nanoemulsion mixed with 1.6×10⁵ PFU RSV strain L19containing 3.3 μg F protein). Cotton rats produced a specific immuneresponse against RSV. The antibody levels were diminished until a secondboost was administered on week 14. There was a slight increase in theantibody levels following the challenge (FIGS. 27 and 28). Inparticular, FIG. 27 shows the serum immune response in the vaccinatedcotton rats. The Y axis shows IgG, μg/mL, over a 14 week period, at day4 post-challenge, and at day 8 post-challenge. FIG. 28 shows the serumimmune response in the vaccinated cotton rats. FIG. 28A shows the endpoint titers (Y axis) over a 14 week period, at day 4 post-challenge,and at day 8 post-challenge. FIG. 28B shows the ELISA units (Y axis)over a 14 week period, at day 4 post-challenge, and at day 8post-challenge.

The efficacy of IM immunization was assessed by challenging the animalswith a live A2 strain of RSV, which is a strain that causes disease inhumans. A dose of 5×10⁵ pfu of RSV strain A2 was administered to animalstwo weeks after booster immunization of the RSV L19nanoemulsion-adjuvanted vaccine. A naïve age-matched group was alsochallenged. Half of the animals in each group were sacrificed on day 4post challenge, at which time the maximum viral load was demonstrated inthe lungs of Cotton Rats. The other half were sacrificed at day 8.

Viral clearance: Lung culture showed that all vaccinated animals had novirus in their lungs at 4 days post challenge while naïve animals hadvirus loads of 10³ pfu RSV strain A2/g of lung tissue (FIG. 21).Specifically, FIG. 29 shows viral clearance at Day 4 in the lungs of thecotton rats, by measurement of the RSV strain A2 viral titer (PFU/g) inthe lungs of the tested cotton rats. Vaccinated cotton rats (vaccinatedwith W₈₀5EC nanoemulsion combined with RSV strain L19), showed completeclearance of RSV strain A2 challenged virus from the lungs of the cottonrats. In contrast, naïve animals showed viral loads of 10³ pfu RSVstrain A2/gram of lung or greater (limit of detection was 2.1×10¹pfu/g).

Cotton Rat Summary: All RSV vaccines formulated in nanoemulsion andadministered IN or IM elicited a protective immune response thatprevented infection of immunized animals. Moreover,nanoemulsion-inactivated and adjuvanted RSV L19 vaccines are highlyimmunogenic in the universally accepted cotton rat model. Cotton ratselicited a rise in antibody titers after one immunization and asignificant boost after the second immunization (approximately a 10-foldincrease). The antibodies generated are highly effective in neutralizinglive virus and there is a linear relationship between neutralization andantibody titers. Furthermore, antibodies generated in cotton rats showedcross protection when immunized with the RSV L19 strain and challengedwith the RSV A2 strain. Both IM and IN immunization established memorythat can be invoked or recalled after an exposure to antigen either as asecond boost or exposure to live virus.

Example 16

The purpose of this example was to compare intranasal (IN) versusintramuscular (IM) administration of a W₈₀5EC nanoemulsion adjuvantedRSV vaccine.

Methods: RSV vaccine containing 2×10⁵ plaque forming units (PFU) of L19RSV virus with 1.7 μg of F protein was inactivated with 20% W₈₀5ECnanoemulsion adjuvant. BALB/C mice (n=10/arm) were vaccinated at weeks 0and 4 IN or IM. Serum was analyzed for anti-F antibodies (FIG. 30).Cells from spleens, cervical and intestinal lymph nodes (LN) wereanalyzed for RSV-specific cytokines (FIG. 31). Mice were challengedoropharyngeally with 5×10⁵ PFU L19 at 8 weeks. Airway hyperreactivitywas assessed by plethysmography. Lungs were analyzed day 8 postchallenge to assess mRNA of cytokines, viral proteins, andhistopathology.

Results: Mice vaccinated IM had higher anti-F antibodies (GM 396 [95%CI-240-652] vs. 2 [95% CI 0-91]) (FIG. 22) and generated more IL-4 andIL-13, after challenge (p<0.05) compared to mice vaccinated IN (FIG.31). In contrast, IL-17 from spleen cells, cervical LN and intestinal LNwas higher after IN vs IM vaccination (GM: 57 vs 1, 119 vs 3 and 51 vs 4pg/mL, respectively, p<0.05) (FIG. 31). FIG. 32 shows measurement of thecytokines IL-4, IL-13, and IL-17 in lung tissue following either IN orIM vaccination. IL-4 and IL-13 were expressed at higher amountsfollowing IM administration, with IL-17 showing greater expressionfollowing IN administration. Upon challenge, both routes of immunizationresulted in clearance of F and G proteins, but airway resistance washigher in the IM group (p=0.03) with confirmatory histopathology (FIG.33). Pulmonary IL-4 and IL-13 had a strong positive correlation (r=0.89;p=0.001 and r=0.8; p=0.007, respectively) with airway hyperreactivity.Pulmonary IL-17 was only generated in mice vaccinated IN (p=0.008) andhad a strong negative correlation (r=−0.81 p=0.007) with airwayhyperreactivity.

Conclusion: Compared to IM vaccination, IN vaccination with a novelnanoemulsion adjuvant W₈₀5EC resulted in less airway hyperreactivity,strongly correlated with high IL-17 production. IL-17 as generated bymucosal vaccination may be an important marker for reduced airwayhyperreactivity in RSV infection.

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1. A vaccine composition comprising: (a) at least one respiratorysyncytial virus (RSV) surface antigen or an antigenic fragment thereof;and (b) an immune-enhancing nanoemulsion or a dilution thereof,comprising droplets having an average size of about 1000 nm or less and:(i) an aqueous phase; (ii) at least one oil; (iii) at least onesurfactant; and (iv) at least one organic solvent; wherein the at leastone RSV surface antigen is comprised within the nanoemulsion.
 2. Thevaccine composition of claim 1, wherein the surface antigen is RSV Fprotein, RSV G protein, an antigenic fragment of RSV F protein, anantigenic fragment of RSV G protein, or any combination thereof.
 3. Thevaccine composition of claim 1, wherein the RSV surface antigen is froma human respiratory syncytial virus deposited with the American TypeCulture Collection (ATCC) as HRSV-L19.
 4. The vaccine composition ofclaim 1, wherein the RSV surface antigen further comprises at least onenucleotide modification denoting attenuating phenotypes.
 5. The vaccinecomposition of claim 1, wherein the RSV surface antigen or an antigenicfragment thereof is present in a fusion protein.
 6. The vaccinecomposition of claim 1, wherein the RSV surface antigen is a peptidefragment of RSV F protein, a peptide fragment of RSV G protein, or anycombination thereof.
 7. The vaccine composition of claim 1, wherein theRSV surface antigen is multivalent.
 8. The composition of claim 7,wherein the multivalent surface antigen is RSV F protein, RSV G protein,an antigenic fragment of RSV F protein, an antigenic fragment of RSV Gprotein, or any combination thereof.
 9. The vaccine composition of claim1, wherein the immune enhancing nanoemulsion is capable of inducing aTh1 immune response, a Th2 immune response, a Th17 immune response, orany combination thereof.
 10. The vaccine composition of claim 1additionally comprising an adjuvant.
 11. The vaccine composition ofclaim 1, further comprising at least one pharmaceutically acceptablecarrier.
 12. The vaccine composition of claim 1, wherein the vaccinecomposition is formulated for administration either parenterally,orally, intranasally, or rectally.
 13. The vaccine composition accordingto claim 12, wherein the parenteral administration is by intradermal,subcutaneous, intraperitoneal or intramuscular injection.
 14. Thevaccine composition of claim 1, further comprising isolated RSV virionparticles.
 15. The vaccine composition of claim 14, wherein the RSVvirion particles are from a human respiratory syncytial virus depositedwith the American Type Culture Collection (ATCC) as HRSV-L19.
 16. Thevaccine composition of claim 14, wherein the RSV virion particles areinactivated by the nanoemulsion.
 17. The vaccine composition of claim14, wherein the RSV viral genome comprises at least one attenuatingmutation.
 18. The vaccine composition of claim 1, wherein the vaccinecomposition: (a) is not systemically toxic to the subject; (b) producesminimal or no inflammation upon administration; or (c) any combinationthereof.
 19. The vaccine composition of claim 1, wherein thenanoemulsion droplets have an average diameter selected from the groupconsisting less than about 1000 nm, less than about 950 nm, less thanabout 900 nm, less than about 850 nm, less than about 800 nm, less thanabout 750 nm, less than about 700 nm, less than about 650 nm, less thanabout 600 nm, less than about 550 nm, less than about 500 nm, less thanabout 450 nm, less than about 400 nm, less than about 350 nm, less thanabout 300 nm, less than about 250 nm, less than about 200 nm, less thanabout 150 nm, less than about 100 nm, greater than about 50 nm, greaterthan about 70 nm, greater than about 125 nm, greater than about 125 nmand less than about 600 nm, and any combination thereof.
 20. The vaccinecomposition of claim 1, wherein the nanoemulsion comprises: (a) anaqueous phase; (b) about 1% oil to about 80% oil; (b) about 0.1% organicsolvent to about 50% organic solvent; and (c) about 0.001% surfactant toabout 10% surfactant.
 21. The vaccine composition of claim 1, whereinthe nanoemulsion comprises: (a) an aqueous phase; (b) about 1% oil toabout 80% oil; (c) about 0.01% organic solvent to about 50% organicsolvent; (d) about 0.001% to about 10% of one or more nonionicsurfactants; and (3) a cationic surfactant.
 22. The vaccine compositionof claim 1, wherein following administration to a subject, the subjectundergoes seroconversion after a single administration of the vaccine.23. A method for inducing an enhanced immunity against diseases causedby respiratory syncytial viruses comprising the step of administering toa subject an effective amount of vaccine comprising: (a) at least oneRSV surface antigen or an antigenic fragment thereof; (b) animmune-enhancing nanoemulsion or a dilution thereof, comprising dropletshaving an average size of about 1000 nm or less and: (i) an aqueousphase; (ii) at least one oil; (iii) at least one surfactant; and (iv) atleast one organic solvent; wherein the RSV surface antigen ispreferentially comprised within the nanoemulsion.
 24. The method ofclaim 23, wherein the surface antigen is RSV F protein, RSV G protein,an antigenic fragment of RSV F protein, an antigenic fragment of RSV Gprotein, or any combination thereof.
 25. The method of claim 24, whereinthe administering comprises parenterally, orally or intranasally. 26.The method of claim 24, wherein the vaccine further comprises isolatedRSV virion particles.
 27. The method of claim 23, wherein the subject isan infant.
 28. The method of claim 23, wherein the subject is elderly,has chronic obstructive pulmonary disease (COPD), is a transplantpatient, or any combination thereof.